Network pharmacology and molecular docking reveal the mechanism of resveratrol in oral squamous cell carcinoma treatment / 口腔疾病防治

Objective@#To explore the molecular mechanism of resveratrol (RES) in the treatment of oral squamous cell carcinoma (OSCC) through the use of biological information methods such as network pharmacology and molecular docking and to provide a theoretical reference for the clinical application of RES in the treatment of OSCC.@*Methods@#The Swiss Target Prediction(http://www.swisstargetprediction.ch), SEA (http://sea.bkslab.org)database, and Pharm mapper database(http://lilab-ecust.cn) were used to retrieve RES-related targets, and the DISGENET (www.disgenet.org), OMIM (https://omim.org) and GeneCards (https://www.genecards.org) databases were used to screen OSCC disease targets. The intersection of drugs and disease targets was determined, and Cytoscape 3.7.2 software was used to construct a "drug-diseasetarget pathway" network. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to construct a target protein interaction network, and the DAVID database was used for enrichment analysis of key proteins. Finally, molecular docking validation of key proteins was performed using AutoDock and PyMOL. The enrichment analysis and molecular docking results were integrated to predict the possible molecular mechanisms of RES treatment in OSCC; western blot was used to determine the effect of resveratrol at different concentrations (50, 100) μmol/L on the expression of Src tyrosine kinase (SRC), epidermal growth factor receptor (EGFR), estrogen receptor gene 1 (ESR1), and phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway proteins in OSCC HSC-3 cells.@*Results@#A total of 243 targets of RES drugs and 6 094 targets of OSCC were identified. A total of 116 potential common targets were obtained by intersecting drugs with disease targets. These potential targets mainly participate in biological processes such as in vivo protein self-phosphorylation, peptide tyrosine phosphorylation, transmembrane receptor protein tyrosine kinase signaling pathway, and positive regulation of RNA polymerase Ⅱ promoter transcription, and they interfere with the PI3K/AKT signaling pathway to exert anti-OSCC effects. The docking results of resveratrol with OSCC molecules indicated that key targets, such as EGFR, ESR1, and SRC, have good binding activity. The results of cell-based experiments showed that resveratrol inhibited the protein expression of SRC, EGFR, ESR1, p-PI3K, and p-AKT in HSC-3 cells in a dose-dependent manner.@*Conclusion@#RES can inhibit the expression of its targets EGFR, ESR1, SRC, p-PI3K, and p-AKT in OSCC cells.

Effect of South African Herb Hoodia gordonii on Liver Glucolipid Metabolism and PI3K/Akt/FoxO1 Signalling Pathway in db/db Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the South African herb Hoodia gordonii （HG） on glucolipid metabolism in diabetic db/db mice and explore the possible mechanisms of HG on the liver of db/db mice based on the phosphoinositide-3 kinase （PI3K）/protein kinase B （Akt）/factor forkhead protein O1 （FoxO1） signaling pathway. MethodA total of 30 db/db mice were randomly divided into five groups according to fasting blood glucose： model group， metformin group （0.195 g·kg-1）， and low dose （0.39 g·kg-1）， medium dose （0.78 g·kg-1）， and high dose （1.56 g·kg-1） HG groups， with six m/m mice in each group， and another six m/m mice were set as normal group. The mice in the normal and model groups were given saline of 9 mL·kg-1 by gavage. Body weight， water intake， and fasting blood glucose of the mice in each group were measured weekly. After six weeks of continuous administration， serum insulin （FINS）， low-density lipoprotein cholesterol （LDL）， total cholesterol （TC）， triglyceride （TG）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， urea， and creatinine （CREA） were measured， and liver sections were embedded and stained with hematoxylin-eosin （HE）， periodic acid-Schiff （PAS）， and oil red O. Protein expression of PI3K p85， p-Akt， and p-FoxO1 in liver was detected by immunohistochemistry. The mRNA expression of PI3K， Akt， and FoxO1 in liver tissue was detected by real-time polymerase chain reaction （Real-time PCR）. ResultAfter six weeks of administration intervention， it was found that fasting blood glucose was significantly downregulated in mice in the three HG groups （P<0.05）. The level of islet resistance index was significantly reduced in both the low and medium dose HG groups （P<0.05）. The expression levels of TC， TG， and LDL were reduced in all HG groups （P<0.05， P<0.01）. Pathologically， HG could alleviate hepatocyte steatosis， reduce the volume and content of lipid droplets in liver， and increase the distribution of glycogen granules in liver to some extent in mice. Immunohistochemical assays revealed that PI3K p85 protein expression was significantly increased in the low， medium， and high dose HG groups compared with the model group （P<0.01）. p-Akt protein expression was significantly increased in the medium and high dose HG groups （P<0.05， P<0.01）. p-FoxO1 protein expression was significantly increased in the low， medium， and high dose HG groups （P<0.05， P<0.01）. Compared with the model group， PI3K mRNA was increased in low dose， medium dose， and high dose HG groups （P<0.05）， and Akt mRNA was increased in high dose HG group （P<0.05）. FoxO1 mRNA was decreased in low dose， medium dose， and high dose HG groups （P<0.05）. ConclusionHG can ameliorate the disorder of glucolipid metabolism in db/db mice， which may be related to its activation of the hepatic PI3K/Akt/FoxO1 signaling pathway.

Analysis of Antidepressant Quality Markers of Bupleuri Radix Before and After Vinegar-processing Based on UPLC-Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.

Effect of Qiwei Baizhusan on Cognitive Dysfunction in Rats with Diabetic Encephalopathy Based on PI3K/Akt/GSK-3β Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the therapeutic effect of Qiwei Baizhusan（QWBZS） on diabetic encephalopathy（DE） rat model， and to explore the possible mechanism of QWBZS in the treatment of DE based on phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）/glycogen synthase kinase-3β（GSK-3β） signaling pathway. MethodForty-eight SPF male Wistar rats were randomly divided into blank group（8 rats） and high-fat diet group（40 rats）. After 12 weeks of feeding， rats in the high-fat diet group were intraperitoneally injected with 35 mg·kg-1 of 1% streptozotocin（STZ） for 2 consecutive days to construct a DE model， and rats in the blank group were injected with the same amount of sodium citrate buffer. After successful modeling， according to blood glucose and body weight， model rats were randomly divided into model group， low， medium and high dose groups of QWBZS（3.15， 6.3， 12.6 g·kg-1）， combined western medicine group（metformin+rosiglitazone， 0.21 g·kg-1）， with 6 rats in each group. The administration group was given the corresponding dose of drug by gavage， and the blank group and the model group were given an equal volume of 0.9% sodium chloride solution by gavage， 1 time/day for 6 weeks. Morris water maze was used to detect the spatial memory ability of DE rats. Fasting insulin （FINS） level was detected by enzyme-linked immunosorbent assay（ELISA） and insulin resistance index（HOMA-IR） was calculated. Hematoxylin-eosin（HE） staining was used to observe the morphological changes of hippocampus in rats， ELISA was used to detect the indexes of oxidative stress in hippocampal tissues， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect mRNA expression levels of PI3K， Akt， nuclear transcription factor-κB（NF-κB）， tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β） in hippocampus， and Western blot was used to detect the protein expression of PI3K， Akt， phosphorylated（p）-Akt， GSK-3β and p-GSK-3β in hippocampus of rats. ResultCompared with the blank group， FINS and HOMA-IR values of the model group were significantly increased（P<0.01）， the path of finding the original position of the platform was significantly increased， and the escape latency was significantly prolonged（P<0.01）， the morphology of neuronal cells in hippocampal tissues was disrupted， the levels of reactive oxygen species（ROS） and malondialdehyde（MDA） in hippocampus of rats were increased， and the activity of superoxide dismutase（SOD） was decreased（P<0.05， P<0.01）， mRNA expression levels of PI3K and Akt were decreased（P<0.01）， mRNA expression levels of NF-κB， TNF-α and IL-1β were increased（P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly decreased， and the protein expression of GSK-3β was significantly increased（P<0.01）. Compared with the model group， the FINS and HOMA-IR values of the medium dose group of QWBZS and the combined western medicine group were significantly decreased（P<0.01）， the path of finding the original position of the platform and the escape latency were significantly shortened（P<0.01）， the hippocampal tissue structure of rats was gradually recovered， and the morphological damage of nerve cells was significantly improved， the contents of ROS and MDA in hippocampus of rats decreased and the level of SOD increased（P<0.01）， the mRNA expression levels of PI3K and Akt were increased（P<0.01）， and the mRNA expression levels of NF-κB， TNF-α and IL-1β were decreased （P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly increased（P<0.01）， and the expression of GSK-3β was significantly decreased（P<0.01）. ConclusionQWBZS can alleviate insulin resistance in DE rats， it may repair hippocampal neuronal damage and improve learning and cognitive ability of DE rats by activating PI3K/Akt/GSK-3β signaling pathway.

Earthworm Protein Protects Vascular Endothelial Function in Spontaneously Hypertensive Rats via PI3K/Akt/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2 （PI3K/Akt/Nrf2） pathway in the aorta of spontaneously hypertensive rats （SHR） and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction （VED）. MethodTen 10-week-old Wistar Kyoto （WKY） rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group， and fifty SHR rats were randomized according to body weight into model， valsartan （8×10-3 g·kg-1·d-1）， and high-， medium-， and low-dose （0.2， 0.1， 0.05 g·kg-1·d-1， respectively） earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period， the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention， enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ （Ang-Ⅱ） and endothelin-1 （ET-1） in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide （NO）， malondialdehyde （MDA）， glutathione peroxidase （GPX）， superoxide dismutase （SOD）， and ferrous ion （Fe2+）. Hematoxylin-eosin （HE） staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of PI3K， Akt， Nrf2， heme oxygenase-1 （HO-1）， and glutathione peroxidase 4 （GPX4） in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the thoracic aorta. ResultCompared with the normal group， the model group had decreased body mass， increased irritability， severe endothelial damage， elevated blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， lowered NO level （P<0.01）， and down-regulated mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the aortic tissue （P<0.01）. Compared with the model group， drug intervention caused no significant change in the body mass， calmed the rats， alleviated the endothelial damage， lowered blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， elevated the NO level （P<0.05）， and up-regulated the mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 （P<0.05）. ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically， it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.

Mechanism Exploration of TG Regulating PI3K/Akt to Improve Insulin Resistance in Liver of T2DM Rats Based on Transcriptomics / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Tangzhi pills on the improvement of insulin resistance （IR） in the liver with type 2 diabetes （T2DM） by regulating phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway based on differential genes and its possible molecular mechanism. MethodT2DM rat models were prepared by high fat （HFD） diet combined with streptozotocin （STZ） intraperitoneal injection. The experiment was divided into blank group， model group， metformin hydrochloride group （0.18 g·kg-1）， Tangzhi pills high （1.08 g·kg-1）， medium （0.54 g·kg-1） and low （0.27 g·kg-1） dose groups. Rat serum， liver， and pancreatic tissue were collected， and the pathological tissue of the liver and pancreas was observed using hematoxylin-eosin （HE） staining. The fasting blood glucose level （FBG） was detected， and oral glucose tolerance （OGTT） tests were conducted. Enzyme-linked immunosorbent assay （ELISA） was used to detect fasting serum insulin （FINS） and glycated hemoglobin （GHb） levels in rats. IR homeostasis model index （HOMA-IR）， β cellular homeostasis index （HOMA-β）， and insulin sensitivity index （ISI） were calculated. Biochemical methods were used to determine the levels of triglyceride （TG）， total cholesterol （TC）， low-density lipoprotein （LDL-C）， and high-density lipoprotein （HDL-C） in rat serum. Transcriptomics obtained differentially expressed mRNA from liver tissue and enriched differentially expressed pathways. Real-time reverse transcriptase polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of cyclic adenylate responsive element binding protein 3-like protein 2 antibody （CREB3l2）， B-lymphocyte tumor 2 （Bcl-2）， Toll-like receptor 2 （TLR2）， cyclin-dependent kinase inhibitor 1A （CDNK1A）， and DNA damage induced transcription factor 4-like protein （DDIT4） in liver tissue. Western blot was used to detect the protein expression of phosphorylated phosphatidylinositol 3-kinase （p-PI3K）， phosphorylated protein kinase B （p-Akt）， glucose transporter 4 （GLUT4）， insulin receptor （INSR）， and insulin receptor substrate 2 （IRS2）. ResultThe pharmacodynamic experiment results showed that compared with model group， Tangzhi pills groups repaired liver and pancreatic tissue to varying degrees， reduced blood sugar （P<0.01）， and promoted a decrease in serum FINS， GHb， and HOMA-IR （P<0.05， P<0.01）. In addition， HOMA-β and ISI increased （P<0.05， P<0.01）. The levels of TC， TG， and LDL-C decreased （P<0.05， P<0.01）， while the levels of HDL-C increased （P<0.05， P<0.01）. The transcriptomics experimental results confirmed that the PI3K/Akt signaling pathway was significantly expressed in both the blank group and model group， as well as in the high-dose Tangzhi pills group and model group. CDNK1A， DDIT4， CREB3l2， Bcl-2， and TLR2 were significantly differentially expressed mRNA during TG intervention in T2DM. Compared with the model group， the protein expression of p-PI3K， p-Akt， GLUT4， INSR， and IRS2 increased in all Tangzhi pills groups （P<0.01）. The mRNA expression of CREB3l2， Bcl-2， and TLR2 increased （P<0.01）， while that of CDNK1A and DDIT4 decreased （P<0.01）. ConclusionTangzhi pills may regulate the PI3K/Akt signaling pathway based on the differential mRNA expression of CREB3l2， Bcl-2， TLR2， CDNK1A， and DDIT4， thereby improving IR in the liver with T2DM.

Modified Tongqiao Huoxuetang Down-regulates PI3K/Akt Pathway to Treat Basilar Artery Dolichoectasia / 中国实验方剂学杂志

ObjectiveTo establish a mouse model of basilar artery dolichoectasia （BAD） and explore the mechanism of modified Tongqiao Huoxuetang （JTQHX） in regulating BAD via phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） pathway. MethodSixty C57/BL6 female mice were randomized into sham operation （injected with 10 U·mL-1 inactivate elastase）， model， atorvastatin calcium tablets （2.6 mg·kg·d-1）， and low- and high-dose （crude drug 3.4， 17 g·kg-1·d-1， respectively） JTQHX groups. The mouse model of BAD was established by injection with 10 U·mL-1 elastase. After 14 days of modeling， the sham operation group and model group were administrated with equal volumes of pure water by gavage， and other groups with corresponding drugs for 2 months. The levels of interleukin-6 （IL-6） and calpain （LpA） in the serum were measured by enzyme-linked immunosorbent assay （ELISA）. Verhoeff 's Van Gieson （EVG） staining was employed to observe the pathological changes of blood vessels. Terminal-deoxynucleotidyl transferase mediated nick end labeling （TUNEL） was employed to examine the apoptosis rate of vascular smooth muscle cells （VSMCs）. Image Pro Plus was used to observe and calculate the curvature index， elongation length， percentage increase in vessel diameter， and curvature angle of the basilar artery vessels in mice. Western blot was employed to determine the expression levels of PI3K and Akt in the vascular tissue. ResultCompared with the sham operation group， the model group showed lowered IL-6 level （P<0.01）， no significant change in LpA level， increased apoptosis of VSMCs （P<0.01）， and increased curvature index， elongation length， percentage increase in vessel diameter， and curvature angle （P<0.01）. Furthermore， the modeling up-regulated the protein levels of PI3K and Akt in blood vessels （P<0.01） and aggravated the destruction of the inner elastic layer， atrophy of the muscular layer， and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall. Compared with the model group， 2 months of treatment with JTQHX elevated the IL-6 level （P<0.01）， reduced the apoptosis of VSMCs （P<0.01）， decreased the curvature index， elongation length， percentage increase in vessel diameter， and curvature angle （P<0.05， P<0.01）， and down-regulated the protein levels of PI3K and Akt in blood vessels （P<0.01）. In addition， the treatment alleviated the destruction of the inner elastic layer， atrophy of the muscular layer， and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall. ConclusionJTQHX inhibits the elongation， expansion， and curvature of basilar artery vessels and alleviates the pathological changes by reducing the apoptosis of VSMCs and down-regulating the expression of PI3K/Akt pathway.

Effect of Stemona tuberosa Alkaloids on Apoptosis and PI3K/Akt and JNK/MAPK Signaling Pathways of Human Lung Cancer A549 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Stemona tuberosa alkaloids （STA） on apoptosis and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase （JNK/p38 MAPK） signaling pathways in human lung cancer A549 cells. MethodA549 cells were classified into blank group and STA groups （100， 150， 200， 250， 300 mg⋅L-1）. Thiazole blue （MTT） assay and colony formation assay were used to evaluate the proliferation of A549 cells. Apoptosis was observed based on Hoechst 33258 staining， flow cytometry， and Annexin V-FITC/PI staining. Western blot was employed to detect the expression of apoptosis-related proteins cysteine-aspartic acid protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， and Bcl-2， and the expression of PI3K， phosphorylated （p）-PI3K， Akt， p-Akt， JNK， p-JNK， p38 MAPK， and p-p38 MAPK. ResultCompared with the blank group， STA groups （150， 200， 250， 300 mg⋅L-1） demonstrated the increase in inhibition rate of cell proliferation （P<0.01） and cell clone inhibition rate， and decrease in cell clone formation rate （P<0.01）. In comparison with the blank group， STA groups showed typical characteristics of apoptosis， such as chromatin condensation and enhanced fluorescence reaction. The apoptosis rate of STA groups was significantly higher than that of the blank group （P<0.01）. Compared with the blank group， STA （150， 200， 250， 300 mg⋅L-1） significantly up-regulated the protein expression of Caspase-3 and Bax （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 protein （P<0.01）. Compared with the blank group， STA had no significant influence on the total protein expression of PI3K， Akt， JNK， and p38 MAPK. However， STA （150， 200， 250， 300 mg⋅L-1） significantly decreased the levels of p-PI3K and p-Akt （P<0.05， P<0.01） and increased the level of p-p38 MAPK （P<0.05， P<0.01）. Compared with the blank group， STA （200， 250， 300 mg⋅L-1） significantly raised the level of p-JNK （P<0.05， P<0.01）. ConclusionSTA can inhibit the proliferation and induce the apoptosis of A549 cells by inhibiting PI3K/Akt signaling pathway and activating JNK/p38 MAPK signaling pathway.

Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Zhuluan decoction on the ovarian reserve function of rats with cyclophosphamide-induced premature ovarian insufficiency， and explore the protective mechanism of Zhuluan decoction in the rat model of premature ovarian insufficiency based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty female SD rats were randomly divided into normal group （n=10） and model group （n=50）. The model group was given intraperitoneal injection of cyclophosphamide （50 mg·kg-1 loading dose on the 1st day+8 mg·kg-1 low-dose maintenance on the 2nd–15th days）. After successfully modeling， the rats were randomly divided into model group， positive drug （progynova） group （0.1 mg·kg-1·d-1）， and low-， medium-， and high-dose Zhuluan decoction groups （14， 28， 56 g·kg-1·d-1 ）， with 10 rats in each group. The model group and the normal group were given equal volume of distilled water by gavage， once a day， continuous administration for 21 d. The estrous cycle and body weight of rats in each group were detected， and the ovarian organ index and uterine organ index were calculated. The ovarian tissue pathology and ovarian follicle counts at all levels were determined by hematoxylin-eosin （HE） staining. The content of the serum antimullerian hormone （AMH）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and inhibin-B （INH-B） of rats was determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of PI3K， Akt， mTOR in the rat ovarian tissue were determined by Western blot. The microtubule-associated protein 1 light chain 3B （LC3B） protein expression in the rat ovarian tissue was determined by immunohistochemistry. ResultAs compared with the blank group， the estrous cycle of rats in the model group was disordered， the body weight， ovarian organ index， and uterine organ index decreased， the number of primordial follicles decreased， and the number of secondary follicles and atretic follicles increased. In the model group， FSH increased （P<0.01）， LH increased （P<0.05）， AMH level decreased （P<0.05）， the protein expression levels of PI3K， Akt， and mTOR in the ovarian tissue decreased （P<0.01）， and the protein expression level of LC3B increased significantly （P<0.01）. As compared with the model group， the above indexes were improved in the progynova group and different doses of Zhuluan decoction groups， the content of AMH increased （P<0.05）， and FSH decreased （P<0.05）. In the progynova group and different doses of Zhuluan decoction groups， the protein expression level of LC3B decreased obviously （P<0.01）， and the protein expression levels of PI3K， Akt， and mTOR all showed an increasing trend. Moreover， there was a statistically significant difference in the progynova group and low- and medium-dose Zhuluan decoction groups （P<0.05）. ConclusionZhuluan decoction may inhibit the occurrence of excessive autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR pathway， thereby reversing the effect of modeling on ovarian reserve in rats.

Mechanism of Buyang Huanwutang Combined with Bone Marrow Mesenchymal Stem Cell Transplantation in Treatment of Spinal Cord Injury Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Buyang Huanwutang combined with bone marrow mesenchymal stem cell （BMSC） transplantation in the treatment of spinal cord injury （SCI）. MethodDifferent concentrations （12.5， 25， 50 g·kg-1） of Buyang Huanwutang were administrated to rats by gavage. The spinal cord function of rats was measured by modified Tarlov score， and the most suitable concentration of Buyang Huanwutang was screened out. SD rats were then divided into 6 groups， namely， the sham operation group （gavage of equal amount of normal saline）， the model group （gavage of equal amount of normal saline）， the Buyang Huanwutang group （gavage of 25 g·kg-1 Buyang Huanwutang）， the BMSC transplantation group （tail vein injection of BMSCs 1 mL）， the Buyang Huanwutang+BMSC group （gavage of 25 g·kg-1 Buyang Huanwutang and tail vein injection of BMSCs 1 mL）， the Buyang Huanwutang+BMSC+LY294002 group （gavage of 25 g·kg-1 Buyang Huanwutang and tail vein injection of BMSCs 1 mL and 40 mg·kg-1 LY294002）， with 10 rats in each group. The spinal cord function was measured by the modified Tarlov score， inclined plate test， and latency of cortical somatosensory evoked potential. Immunohistochemistry was used to detect the number of 5-bromo-2-deoxyuracil nucleoside （Brdu）-labeled positive cells in the spinal cord tissue. The protein expression levels of phosphorylated protein kinase B （p-Akt）， glycoprotein 130 （gp130）， and interleukin-6 （IL-6） in spinal cord were detected by Western blot. ResultAs compared with the sham operation group， the Tarlov score and the critical angle of tilt plane in the model group were significantly decreased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 were significantly increased （P<0.05）. As compared with the model group， the Tarlov score and the critical angle of tilt plane in the sham operation group and each treatment group were significantly increased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 were significantly decreased （P<0.05）. As compared with the BMSC group， the Tarlov score and the critical angle of inclined plane in the Buyang Huanwutang+BMSC group increased （P<0.05）， the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 decreased （P<0.05）， and the number of Brdu-labeled positive cells increased 5 weeks after transplantation （P<0.05）. As compared with the Buyang Huanwutang+BMSC group， the Tarlov score and the critical angle of the inclined plane in the Buyang Huanwutang+BMSC+LY294002 group increased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 decreased significantly （P<0.05）. Five weeks after transplantation， the number of Brdu-labeled positive cells increased significantly in the Buyang Huanwutang+BMSC+LY294002 group （P<0.05）. ConclusionBuyang Huanwutang can promote BMSCs migration and restore spinal cord function by inhibiting phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signal.

Effect and Mechanism of Osthole on Proliferation and Apoptosis in Human Intrahepatic Cholangiocarcinoma HuCCT1 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of osthole on the proliferation and apoptosis in human intrahepatic cholangiocarcinoma HuCCT1 cells. MethodThe effect of 10， 20， 40， 80， and 120 μmol·L-1 osthole on the proliferation of HuCCT1 cells was detected by the cell counting kit-8 （CCK-8）. A blank group， and low-， medium-， and high-dose osthole groups （16， 32， and 64 μmol·L-1） were set up. The effect of osthole on cell clone formation rate was detected by colony formation assay. The effect of osthole on cell cycle and apoptosis was detected by flow cytometry. The effect of osthole on cell apoptotic morphology was detected by Hoechst 33342 fluorescent staining. The effect of osthole on cell cycle protein cyclin B1， proliferating cell nuclear antigen （PCNA）， cysteine-aspartic acid protease （Caspase）-9， Caspase-3， cleaved Caspase-9， cleaved Caspase-3， cleaved poly（ADP-ribose） polymerase （cleaved PARP）， B-cell lymphoma-2 （Bcl-2）， phosphorylated protein kinase B （p-Akt）， phosphorylated mammalian target of rapamycin （p-mTOR）， and phosphorylated ribosomal protein S6 （p-RPS6） was detected by Western blot. ResultThe cell viability in the osthole group（40，80，120 μmol·L-1） decreased （P<0.05，P<0.01）， with the half maximal inhibitory concentration （IC50） of 63.8 μmol·L-1 as compared with that in the blank group. Compared with the blank group， the osthole groups（32，64 μmol·L-1）showed reduced clone formation rate （P<0.01）， increased number of cells in the G2 phase （P<0.05，P<0.01）， decreased number of cells， increased pyknosis and fragmentation， increased apoptosis rate （P<0.05，P<0.01）， down-regulated expression of cyclin B1， PCNA， Bcl-2， Caspase-3， Caspase-9， p-Akt， p-mTOR， and p-RPS6 （P<0.05，P<0.01）， and up-regulated expression of cleaved Caspase-3， cleaved Caspase-9， and cleaved PARP （P<0.05，P<0.01）. ConclusionOsthole can inhibit the proliferation and promote the apoptosis of HuCCT1 cells， and its mechanism may be related to the Akt/mTOR signaling pathway.

Mechanism of curcumin inhibiting choroidal neovascularization in brown Norway rat / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the mechanism of curcumin inhibiting the choroidal neovascularization(CNV)of brown Norway(BN)rats.METHODS: CNV model of 36 BN rats was established through laser photocoagulation induction, and they were divided into 6 groups with 6 rats in each group. Normal group was fed normally with no intervention, while 532nm laser photocoagulation was used to establish a experimental CNV model in BN rats. Rats after modeling were respectively intervened for 14d and divided into model group, ranibizumab group, curcumin low [100mg/(kg·d)], medium [200mg/(kg·d)], and high [400mg/(kg·d)] dose group. The model group was given intragastric administration of saline for 14d, ranibizumab(10mg/mL, 0.2mL/dose)was injected at 2d after photocoagulation with 5μL once for rats in ranibizumab group, and different concentrations of curcumin were intragastrically administrated to the rats in low, medium and high groups for 14d. Fundus photography, fundus fluorescein angiography(FFA)and indocyanine green angiography(ICGA)examination were performed at 14d after photocoagulation. Ocular histopathological specimens of rats with CNV were made, and the central thickness of CNV were observed by HE staining. Ocular histopathological specimens were made, and the expressions of AKT/p-AKT/HIF-1α/VEGF signaling pathway-related proteins were observed by immunohistochemistry. The mRNA relative expressions of AKT/HIF-1α/VEGF factor in CNV tissues were detected by RT-qPCR, and the protein expressions of AKT/p-AKT/HIF-1α/VEGF factor in CNV tissues were detected by Western-blot.RESULTS: CNV generation rates in the model group, the ranibizumab group, and the low, medium and high-dose curcumin groups were 78.18%, 73.21%, 77.19%, 75.86%, 74.55%, respectively, which were higher than 70%. The average absorbance were 182.12±6.59, 119.22±8.03, 166.45±8.33, 164.34±5.69, 149.22±6.45, respectively; the ranibizumab group was significantly lower than the model group(P&#x0026;#x003C;0.05); the low-dose, medium-dose and high-dose groups were significantly higher than the ranibizumab group(P&#x0026;#x003C;0.05), and the curcumin high-dose group was significantly lower than the model group(P&#x0026;#x003C;0.05). HE staining showed that the retinal tissue structure of BN rats in normal group was clear and neatly arranged. The central thickness of CNV in the ranibizumab group was significantly reduced at 14d after photocoagulation compared with the model group(P&#x0026;#x003C;0.05); While the curcumin high-dose group was significantly reduced compared with the model group(P&#x0026;#x003C;0.05), but increased when compared with ranibizumab group(P&#x0026;#x003C;0.05). Immunohistochemistry results showed that AKT, p-AKT, HIF-1α, and VEGF factors were negatively expressed in the retinal tissue structure of BN rats in the normal group, and no brown-yellow reactants were found. The expression of AKT, p-AKT, HIF-1α, and VEGF factors in the model group were higher than that in the normal group at 14d after photocoagulation(P&#x0026;#x003C;0.05); the ranibizumab group was lower than the model group(P&#x0026;#x003C;0.05). While the expression of the curcumin high-dose group was significantly decreased compared with the model group(P&#x0026;#x003C;0.05), but significantly increased when compared with ranibizumab group(P&#x0026;#x003C;0.05). The mRNA results showed that the relative expression levels of AKT, HIF-1α and VEGF mRNA in the model group at 14d after photocoagulation were higher than those of the normal group(P&#x0026;#x003C;0.05); the ranibizumab group was lower than the model group(P&#x0026;#x003C;0.05). While curcumin high-dose group was significantly decreased compared with the model group(P&#x0026;#x003C;0.05), but significantly increased when compared with ranibizumab group(P&#x0026;#x003C;0.05). Western-blot results showed that there was no significant difference in the relative expression of AKT protein among each experimental groups at 14d after photocoagulation. The relative expression of p-AKT protein in the model group was significantly higher than that in the normal group(P&#x0026;#x003C;0.05); the ranibizumab group was significantly lower than the model group(P&#x0026;#x003C;0.05); the curcumin high-dose group was significantly lower than the model group(P&#x0026;#x003C;0.05). The relative expression levels of HIF-1α protein were significantly higher in the model group than in the normal group(P&#x0026;#x003C;0.05), and the ranibizumab group was lower than in the model group(P&#x0026;#x003C;0.05). The relative expression levels of HIF-1α protein was lower in the curcumin high-dose group than in the model group(P&#x0026;#x003C;0.05)but higher than ranibizumab group(P&#x0026;#x003C;0.05). The relative expression level of VEGF protein was significantly lower in the curcumin medium/high-dose group than in the model group(P&#x0026;#x003C;0.05).CONCLUSION: Curcumin at 400mg/(kg·d)has an inhibitory effect on CNV in BN rats. The mechanism may be closely related to inhibiting the activation of AKT/p-AKT/HIF-1α/VEGF signaling pathways.

Effect of xenon post-conditioning on spinal cord ischemia/reperfusion injury in rats: regulating Akt signaling pathway and autophagy / 中国康复理论与实践

ObjectiveTo investigate the effect of xenon post-conditioning on autophagy after spinal cord ischemia/reperfusion injury (SCIRI) in rats and its relationship with protein kinase B (Akt) signaling pathway. MethodsA total of 30 male rats were randomized into sham-operated group (sham group), spinal cord ischemia/reperfusion injury group (I/R group) and I/R + xenon post-conditioning group (Xe group), with ten rats in each group. In the latter two groups, SCIRI was induced by clamping the abdominal aorta for 85 minutes followed by reperfusion for four hours. Xe group inhaled xenon and oxygen (1∶1) for one hour at one hour after initiation of reperfusion, while the other groups inhaled nitrogen and oxygen (1∶1) for one hour. After the reperfusion, they were assessed with Basso-Beattie-Bresnahan (BBB) scale and slanting board test. And then, their spinal cords of L3-5 were obtained. Nissl staining was used to count the number of normal neurons. Western blotting was used to detect the protein expression of Akt, p-Akt, p62, Beclin 1, microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3 Ⅱ. The mRNA expression of Beclin 1, p62 and LC3 Ⅱ in the spinal cord was measured with reverse transcription real-time quantitative polymerase chain reaction. ResultsCompared with the sham group, the BBB score and the maximum inclination of the slanting board test decreased, the count of normal neurons decreased, the protein expression of p62 and the p-Akt/Akt ratio decreased (P < 0.01), the protein and mRNA expression of Beclin 1 and LC3 Ⅱ, and the LC3 Ⅱ/LC3 Ⅰ ratio increased, the p62 mRNA expression decreased (P < 0.01) in the I/R group. Compared with the I/R group, the BBB score and the maximum inclination of the slanting board test increased, the count of normal neurons increased, the protein expression of p-Akt and p62 increased, the p-Akt/Akt ratio increased, the protein and mRNA expression of Beclin 1, LC3 Ⅱ and LC3 Ⅱ/LC3 Ⅰ ratio decreased, and the mRNA expression of p62 increased (P < 0.01) in Xe group. ConclusionXenon post-conditioning may relieve SCIRI in rats, which is related to activating Akt signaling pathway to inhibit autophagy.

Repair Mechanism of Jianpi Huogu Prescription on Vascular Injury in Alcohol-induced Osteonecrosis of Femoral Head Based on VEGF/VEGFR2/PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

MethodIn the experiment， 46% vol Red Star Erguotou （10 mL·kg·d-1） was used to establish the AONFH rat model， and the intervention effect of JPHGP at different doses （2.5， 5.0， 10.0 g·kg-1） was observed. Jiangusheng pill （JGS， 1.53 g·kg-1） was selected as the positive control. After 8 weeks of administration， the bone histomorphometry of the femoral head was analyzed by Micro-CT imaging， and the area of medullary microvessels in the femoral head was detected by ink perfusion. The pathological change was observed by hematoxylin and eosin （HE） staining. The protein expressions of Platelet endothelial cell adhesion molecule-1 （CD31）， VEGF， VEGFR2， PI3K， phosphor-Akt （p-Akt） and phosphatase and Tensin homologue deleted on chromosome 10 （PTEN） in the femoral head were determined by immunohistochemistry and Western blot. ResultCompared with normal group， the model group presented the fracture and thinning of trabeculae in the femoral head， increased empty bone lacunae， and elevated number and diameter of adipocytes （P<0.01）. Micro-CT imaging revealed a decrease in bone mineral density （BMD）， bone volume fraction （BV/TV）， trabecular thickness （Tb.Th） and trabecular number （Tb.N） （P<0.05， P<0.01） while an increase in bone surface-to-volume ratio （BS/BV） and trabecular separation （Tb.Sp） （P<0.01）. The results of ink perfusion showed that the area of medullary microvessels in the femoral head was reduced （P<0.01）. Compared with model group， JPHGP lowered the empty bone lacunae rate as well as the number and diameter of adipocytes in the femoral head of AONFH rats. Micro-CT imaging indicated that JPHGP low-dose group had elevated BV/TV， Tb.Th and Tb.N （P<0.05， P<0.01） while decreased BS/BV （P<0.01）， and there was an upward trend in BMD while a downward trend in Tb.Sp， but without statistical difference. In addition， JPHGP medium- and high-dose groups had a rise in BMD， BV/TV， Tb.Th and Tb.N （P<0.05， P<0.01）， a decrease in BS/BV and Tb.Sp （P<0.05， P<0.01） and enlarged area of medullary microvessels in the femoral head （P<0.05， P<0.01）. The expressions of CD31， VEGF， VEGFR2， PI3K， p-Akt in the model group were lower than those in the normal group （P<0.01）， and after medium and high doses of JPHGP treatment， the expressions of CD31， PI3K and p-Akt in the femoral head of rats were up-regulated （P<0.01） while the protein expression of PTEN was down-regulated （P<0.01）. Moreover， JPHGP up-regulated the expressions of VEGF and VEGFR2 （P<0.05， P<0.01）. ConclusionJPHGP can repair the vascular injury in AONFH， and its mechanism may be related to the activation of VEGF/VEGFR2/PI3K/Akt signaling pathway. This study provides certain scientific basis and reference for the clinical application of JPHGP. ObjecctiveTo observe the repair effect of Jianpi Huogu prescription （JPHGP） on vascular injury in experimental alcohol-induced osteonecrosis of femoral head （AONFH）， and to explore its mechanism based on vascular endothelial growth factor （VEGF）/VEGFR2/phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway.

Mechanism of Dihuang Yinzi in Improving Astrocyte Injury and Glycolysis in AD Mice via PI3K/Akt Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Dihuang Yinzi in improving astrocyte injury and glycolysis in Alzheimer's disease （AD） mice via regulating the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， thereby improving the cognitive function of AD mice. MethodForty male APP/PS1 transgenic mice aged four months were randomly divided into a model group and a model + Dihuang Yinzi （0.25 g·kg-1） group， with 20 mice in each group. Forty C57BL/6J mice with the same background and same age were randomly divided into a control group and a control + Dihuang Yinzi （0.25 g·kg-1） group， with 20 mice in each group. The mice in the control + Dihuang Yinzi group and the model + Dihuang Yinzi group were administered with Dihuang Yinzi by gavage， and those in the control group and the model group received an equal volume of sterilized normal saline， once a day for 150 days. Morris water maze test was performed to test the ability of navigation and space exploration of mice. The protein expression of p-PI3K， PI3K， p-Akt， Akt， phosphofructokinase-1 （PFK-1）， and aldehyde dehydrogenase 3 family member B2 （ALDH3B2） in mouse brain tissues was measured by Western blot. An immunofluorescence assay was performed to detect astrocyte morphology and the expression level of ALDH3B2. ResultAs compared with the control group， the model group showed prolonged escape latency during the 2nd to 5th days of the location-based navigation （P<0.05， P<0.01）， reduced number of times crossing the target area of the platform， shortened residence time in the target quadrant （P<0.05， P<0.01）， prolonged residence time in the opposite quadrant （P<0.05）， increased surface area of the cell body and total length of cell protrusions of astrocytes （P<0.05， P<0.01）， and down-regulated protein expression of p-PI3K， p-Akt， ALDH3B2， and PFK-1 （P<0.01）， while the above experimental indexes were not significantly different in the control + Dihuang Yinzi group. Compared with the model group， the model + Dihuang Yinzi group showed shortened escape latency of APP/PS1 mice during the 2nd to 5th days of the location-based navigation （P<0.05， P<0.01）， increased number of times crossing the platform， prolonged target quadrant residence time （P<0.05， P<0.01）， shortened residence time in the opposite quadrant （P<0.05）， reduced surface area of the cell body and total length of cell protrusions of astrocytes （P<0.05）， and up-regulated protein expression of p-PI3K， p-Akt， ALDH3B2， and PFK-1 （P<0.01）. ConclusionDihuang Yinzi can improve the learning and memory ability of AD mice by activating the PI3K/Akt signaling pathway and up-regulating the protein expression of PFK-1 and ALDH3B2 to protect against astrocyte injury in brain tissues and improve glycolysis.

Ganoderma lucidum Polysaccharides Inhibit Malignant Phenotype of Hepatocellular Carcinoma Cells by Regulating PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Ganoderma lucidum polysaccharides （GLP） on the proliferation， migration， cycle， and apoptosis of hepatocellular carcinoma SK­HEP­1 and Huh­7 cells and to explore the underlying mechanism. MethodSK-HEP-1 and Huh-7 cells were classified into the blank group and low-， medium-， and high-dose GLP groups （3.5， 7， 14 g·L-1）. The proliferation of the cells was examined by cell counting kit-8 （CCK­8） assay， and the migration by scratch assay. Cell cycle was measured by flow cytometry and apoptosis was detected based on Hoechst33258 staining. In addition， the expression of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated PI3K （p­PI3K）， and phosphorylated Akt （p­Akt） in the cells was determined by Western blot. ResultCompared with the blank group， the three doses of GLP reduced the proliferation and migration of SK­HEP­1 and Huh­7 cells （P<0.05）， increased the percentage of cells in G1 phase （P<0.05）， and decreased percentage of cells in S and G2 phase （P<0.05）. In addition， the three doses can induce apoptosis of both SK-HEP-1 and Huh-7 cells， particularly the high dose. Moreover， the three doses of GLP lowered the levels of p­PI3K and p­Akt （P<0.05）. ConclusionGLP significantly inhibited the malignant phenotype of SK-HEP-1 and Huh-7 cells through the PI3K/Akt signaling pathway.

Effect of Baihu Jia Renshen Tang on PI3K/Akt Signaling Pathway in Liver of MKR Diabetic Mice / 中国实验方剂学杂志

ObjectiveTo explore the effects of Baihu Jia Renshen Tang （BHRS） on the related molecules on the phosphatidylinositol-3-kinase/protein kinase B（PI3K/Akt）signaling pathway in the liver of MKR diabetic model mice. MethodThirty 6-week-old MKR mice were selected and fed on a high-fat diet for four weeks，followed by intraperitoneal injection of streptozotocin（STZ）for the diabetes model establishment. The model was properly induced in the case of the fasting blood glucose （FBG） of ≥11.1 mmol·L-1. After modeling，the mice were randomly divided into a model group，a BHRS group （12.09 g·kg-1·d-1），and a metformin group （0.065 g·kg-1·d-1），with 10 mice in each group. Ten FVB mice were assigned to the control group. The mice in the groups with drug intervention were continuously administered correspondingly for 28 days. After administration，the mice were sacrificed，followed by the oral glucose tolerance test （OGTT） and FBG detection. Serum very low-density lipoprotein（VLDL）content was determined by semi-quantitative enzyme-linked immunosorbent assay （ELISA）. Four indexes related to blood lipid were determined by the biochemistry analyzer. Liver tissues were subjected to pathological examination by hematoxylin-eosin（HE）staining. Western blot was used to detect the protein expression of PI3K，Akt，phosphorylated（p）-PI3K，p-Akt，forkhead box protein O1 （FoxO1），insulin receptor（InsR），and insulin receptor substrate-2（IRS-2） in liver tissues of mice. Real-time polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression of PI3K，Akt，FoxO1，InsR，and IRS-2 in liver tissues of mice. ResultCompared with the control group，the model group showed poor general conditions，abnormal glucose tolerance （P<0.05），increased FBG （P<0.01），abnormal blood lipid metabolism，increased serum total cholesterol （TC），triglyceride（TG），low-density lipoprotein cholesterol（LDL-C），and VLDL （P<0.05），decreased level of high-density lipoprotein cholesterol（HDL-C）（P<0.05），fatty degeneration and obvious pathological changes of liver cells，reduced protein expression of PI3K，Akt，p-PI3K/PI3K，p-Akt/Akt，IRS-2，and InsR in liver tissues（P<0.05），increased protein expression of FoxO1（P<0.05），decreased mRNA expression of PI3K，Akt，IRS-2，and InsR in liver tissues （P<0.05），and increased FoxO1 mRNA expression（P<0.05）. Compared with the model group，the BHRS group showed improved general conditions and glucose and lipid metabolism （P<0.05），improved pathological state of liver cells，increased protein expression of PI3K，Akt，p-PI3K/PI3K，p-Akt/Akt，IRS-2，and InsR in liver tissues（P<0.05），decreased protein expression of FoxO1（P<0.05），increased mRNA expression of PI3K，Akt，IRS-2，and InsR in liver tissues （P<0.05），and reduced FoxO1 mRNA expression（P<0.05）. ConclusionBHRS can effectively reduce blood glucose，regulate blood lipid metabolism，and improve the pathological state of the liver in MKR diabetic mice，and its mechanism of action may be related to the regulation of the activity of molecules on the PI3K/Akt signaling pathway.

Effect of hydrogen-rich water on brain injury in rats with acute radiation based on PI3K/AKT/Caspase-9 signaling pathway / 环境与职业医学

Background At present, radiation therapy is widely used in clinical treatment of tumors. However, while radiation therapy damages tumor cells, it also injures surrounding normal tissues. Studies have shown that hydrogen is a potential radiation-protective agent. Objective To investigate the neuroprotective mechanisms of hydrogen-rich water activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/cysteinyl aspartate specificproteinase-9 (Caspase-9) signaling pathway in acute radiation-induced brain injury. Methods Forty male SD rats were randomly divided into four groups: control group, irradiation only group (IR), high-dose hydrogen-rich water intervention group (IR+HHRW), and low-dose hydrogen-rich water intervention group (IR+LHRW), 10 rats in each group. Except for the control group, animals in each group received a single 20 Gy whole brain irradiation. Animals in all groups were gavaged once a day from 3 d before irradiation to 7 d after irradiation, pure water (20 mL·kg−1) was given to the control and the IR groups, and hydrogen-rich water (20 mL·kg−1, 10 mL·kg−1) was given to the IR+HHRW and the IR+LHRW groups. After 7 d of intervention, 5 rats in each group were selected for the Morris water maze experiment for behavioral evaluation. Autopsies were conducted after anesthesia for the remaining animals and blood samples were collected for hematological analysis. Rat brains were harvested for TUNEL staining to observe neuronal apoptosis. HE staining was performed to observe histopathological changes, enzyme-linked immunosorbent assay was adopted to detect oxidative stress-related indicators, and real-time PCR and Western blotting were used to measure the expressions of PI3K/AKT/Caspase-9 pathway-related genes and proteins. Results The body weight of rats receiving irradiation decreased after 7 d of irradiation compared with the control group (P<0.05), and the symptoms such as arched back and malaise occurred to varying degrees, and the symptoms of rats in the IR+HHRW group were significantly milder than those in the IR group. The behavioral test results showed that the escape latency of rats in the IR+HHRW group or the IR+LHRW group was shorter than that in the IR group from day 2 to day 5 (P<0.05), and it took less time for rats in the IR+HHRW group to reach the original position after removing the platform on day 6 (P<0.05). The hematological test results showed that red blood cell (RBC) count, hemoglobin (HGB) level, and white blood cell (WBC) count were significantly decreased in the IR group (P<0.05), and the changes in the IR+HHRW group were improved (P<0.05). The HE staining results showed that the number of abnormal nerve cells, broken and dissolved nuclei, and the degree of damage in the IR+HHRW group were significantly reduced than those in the IR group. The results of oxidative stress evaluation showed that the ability of the IR group to inhibit free radicals decreased, the level of malondialdehyde (MDA) increased (P<0.01); the MDA level decreased after LHRW intervention (P<0.05); the SOD activity was elevated after HHRW intervention (P<0.05). The TUNEL staining results showed that the apoptosis signals in the IR+HHRW group were sparser than those in the IR group (P<0.05). The real-time PCR results showed that compared with the IR group, the mRNA expression levels of PI3K and AKT in the IR+HHRW group and the IR+LHRW group increased (P<0.05), while the mRNA expression levels of Cytc and Caspase-9 decreased (P<0.05). The Western blotting results showed that compared with the IR group, the phospho-AKT (pAKT) protein expression level in the IR+HHRW group increased significantly (P<0.05), while the expression of Caspase-9 and Cytc proteins decreased significantly (P<0.05). Conclusion Hydrogen-rich water can significantly reduce inflammation and oxidative stress caused by acute irradiation-induced brain injury, and decrease neuronal apoptosis. The mechanism may be related to the PI3K/AKT/Caspase-9 signaling pathway.

Renshen Baidusan Combined with Yurui Enema Treats Intestinal Mucosal Injury in Rat Model of Ulcerative Colitis via PI3K/Akt/NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanisms of internal treatment （Renshen Baidusan）， external treatment （Yurui Enema）， and combination of the two methods in treating intestinal mucosal injury in the rat model of ulcerative colitis （UC） from the changes of phosphatidylinositol-3 kinase （PI3K）/protein kinase B （Akt）/nuclear factor-κB （NF-κB） pathway. MethodFifty SPF-grade SD rats were randomized into blank， model， Renshen Baidusan （15.6 g·kg-1）， Yurui Enema （25 g·kg-1）， and combined treatment （15.6 g·kg-1 Renshen Baidusan + 25 g·kg-1 Yurui Enema） groups （n=10）. The rat model of UC was established in other groups except the blank group by 2，4， 6-trinitrosulfonic acid （TNBS）/ethanol. The rats were administered with corresponding drugs once a day for 14 consecutive days since the 8th day after modeling. The histopathological changes of colon were observed by hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of tumor necrosis factor （TNF）-α， interferon （IFN）-γ， interleukin （IL）-4， and IL-10 in the colon tissue. The apoptosis of colon epithelial cells was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling （TUNEL）. The location and expression of B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， TNF-α， and IL-6 in the colon tissue were examined by immunohistochemistry. Real-time quantitative fluorescence polymerase chain reaction （Real-time PCR） and Western blot were employed to determine the mRNA and protein levels， respectively， of the proteins in the PI3K/Akt/NF-κB pathway in the colon tissue. ResultIn the model group， HE staining showed a large number of inflammatory cell infiltration in the mucosa and submucosa. Compared with the blank group， the model group showed elevated levels of TNF-α and IFN-γ and lowered levels of IL-4 and IL-10 in the colon tissue， increased apoptosis rate of colon epithelial cells， increased positive expression of Bax， TNF-α， and IL-6， and decreased positive expression of Bcl-2 （P<0.05）. Moreover， the model group showed up-regulated mRNA levels of PI3K， Akt， and NF-κB and protein levels of PI3K， p-PI3K， Akt， p-Akt， p65， p-p65， Bax， and cleaved Caspase-3， increased Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 ratios， and down-regulated protein levels of NF-κB suppressor protein α（IκBα）， Bcl-2， and Caspase-3 in the colon tissue （P<0.05）. Compared with the model group， the internal treatment， the external treatment， and the combination （referred to as the three groups） alleviated the colonic mucosal injury， lowered the levels of TNF-α and IFN-γ and elevated the levels of IL-4 and IL-10 in the colon tissue， decreased the apoptosis rate of colon cells， inhibited the positive expression of Bax， TNF-α， and IL-6， and promoted the positive expression of Bcl-2 （P<0.05）. Furthermore， the combination group down-regulated the mRNA level of PI3K （P<0.05）. The three groups down-regulated the mRNA levels of Akt and NF-κB and the protein levels of p-PI3K， Akt， p-Akt， p65， p-p65， Bax， and cleaved Caspase-3 in the colon tissue， decreased the Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 ratios， and up-regulated the protein levels of IκBα， Bcl-2， and Caspase-3 （P<0.05）. ConclusionRenshen Baidusan， Yurui Enema， and their combination may inhibit the activation of PI3K/Akt/NF-κB signaling pathway and regulate the expression of genes and proteins related to this pathway to achieve anti-inflammatory and anti-apoptotic effects， thus restoring the intestinal mucosal barrier function of UC rats.

Effect of electroacupuncture on liver Akt/FoxO1 signaling pathway in rats with diabetic fatty / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) on liver protein kinase B (Akt)/forkhead box transcription factor 1 (FoxO1) signaling pathway in Zucker diabetic fatty (ZDF) rats, and to explore the possible mechanism of EA on improving liver insulin resistance of type 2 diabetes mellitus.@*METHODS@#Twelve male 2-month-old ZDF rats were fed with high-fat diet for 4 weeks to establish diabetes model. After modeling, the rats were randomly divided into a model group and an EA group, with 6 rats in each group. In addition, six male Zucker lean (ZL) rats were used as the blank group. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6), "Weiwanxiashu" (EX-B 3), and "Pishu" (BL 20). The ipsilateral "Zusanli" (ST 36) and "Weiwanxiashu" (EX-B 3) were connected to EA device, continuous wave, frequency of 15 Hz, 20 min each time, once a day, six times a week, for a total of 4 weeks. The fasting blood glucose (FBG) in each group was compared before modeling, before intervention and after intervention; the serum levels of insulin (INS) and C-peptide were measured by radioimmunoassay method, and the insulin resistance index (HOMA-IR) was calculated; HE staining method was used to observe the liver tissue morphology; Western blot method was used to detect the protein expression of Akt, FoxO1 and phosphoenolpyruvate carboxykinase (PEPCK) in the liver.@*RESULTS@#Before intervention, compared with the blank group, FBG was increased in the model group and the EA group (P<0.01); after intervention, compared with the model group, FBG in the EA group was decreased (P<0.01). Compared with the blank group, the serum levels of INS and C-peptide, HOMA-IR, and the protein expression of hepatic FoxO1 and PEPCK were increased (P<0.01), while the protein expression of hepatic Akt was decreased (P<0.01) in the model group. Compared with the model group, the serum levels of INS and C-peptide, HOMA-IR, and the protein expression of hepatic FoxO1 and PEPCK were decreased (P<0.01), while the protein expression of hepatic Akt was increased (P<0.01) in the EA group. In the model group, the hepatocytes were structurally disordered and randomly arranged, with a large number of lipid vacuoles in the cytoplasm. In the EA group, the morphology of hepatocytes tended to be normal and lipid vacuoles were decreased.@*CONCLUSION@#EA could reduce FBG and HOMA-IR in ZDF rats, improve liver insulin resistance, which may be related to regulating Akt/FoxO1 signaling pathway.