GSK3B inhibition reduced cervical cancer cell proliferation and migration by modulating the PI3K/Akt signaling pathway and epithelial-to-mesenchymal transition

Previous studies show that glycogen synthase kinase 3β (GSK3B) plays an important role in tumorigenesis. However, its role in cervical cancer is unclear. The present study silenced GSK3B with siRNAs and/or chemical inhibitors to determine its role in HeLa cervical cancer cell proliferation and migration as well as in xenograft tumor growth. Cell Counting Kit (CCK)-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to determine cell survival and proliferation. Scratch and Transwell® assays were used to evaluate cell migration. Xenograft tumors were used to evaluate the effect of GSK3B on tumor growth. Transcriptomic sequencing was used to clarify the mechanisms underlying the foregoing processes. Public databases and clinical specimens showed that GSK3B was upregulated in cervical cancer tissues and correlated with poor prognosis. In vitro experiments indicated that GSK3B inhibition reduced cell viability, proliferation, and migration. In vivo experiments demonstrated that GSK3B inhibition slowed xenograft tumor growth. Transcriptomic sequencing revealed that GSK3B inhibition modulated the phosphatidylinositol 3-carboxykinase (PI3K)/protein kinase B (Akt) and extracellular matrix (ECM)-receptor interaction signaling pathways. GSK3B inhibition decreased the protein levels of phosphorylated PI3K and Akt and the levels of mesenchymal markers but increased those of epithelial markers. An activator of the PI3K/Akt signaling pathway counteracted the suppressive effects of GSK3B inhibition on HeLa cell viability and proliferation and on PI3K/Akt signaling. Our data suggested that GSK3B regulated cervical cancer cell proliferation and migration by modulating the PI3K/Akt signaling pathway and epithelial-to-mesenchymal transition (EMT).

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.

Inhibitory Effect of Sesquiterpenoid M36 from Myrrha on Growth of Human Hepatoma Cells / 中国实验方剂学杂志

ObjectiveThe antitumor activity of sesquiterpenoid M36 isolated from Myrrha against human hepatoma HepG2 cells was investigated in this study. MethodHepG2 cells were treated with M36 at different concentrations （0， 2， 4， 6， 8， 10 μmol·L-1）. Firstly， the effects of M36 on the proliferation of human hepatoma HepG2 cells were detected by methyl thiazolyl tetrazolium （MTT）， colony formation assay， and EdU proliferation assay. Hoechst staining， flow cytometry analysis， and Western blot were used to explore the effect of M36 on the apoptosis of human hepatoma HepG2 cells. Acridine orange staining and western blotting were used to examine the effect of M36 on autophagy in HepG2 cells. Finally， Western blot was used to detect protein expression of cancer-related signaling pathways. ResultCompared with the blank group， M36 treatment significantly inhibited the proliferation of human hepatoma HepG2 cells （P<0.01）， and the half inhibitory concentration （IC50） value of M36 for 48 h was 5.03 μmol·L-1， in a dose- and time-dependent manner. M36 was also able to induce apoptosis and autophagy in human hepatoma HepG2 cells. After treatment with 8 μmol·L-1 M36 for 48 hours， the apoptosis rate of HepG2 cells was （42.03±9.65）% （P<0.01）. Compared with the blank group， HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h had a significant increase in cleaved poly ADP-ribose polymerase （cleaved-PARP） protein levels （P<0.01）. Acridine orange staining showed that autophagy was significantly activated in HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h compared with the blank group （P<0.01）， which was further verified by the up-regulation of microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. Western blot results showed that compared with the blank group， the levels of phosphorylated extracellular regulated protein kinase （p-ERK）， phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK）， phosphorylated c-Jun N-terminal kinase （p-JNK）， and its downstream nuclear transcription factors c-Jun and p-c-Jun protein were significantly increased in M36 group （P<0.05， P<0.01）. The mechanism may be related to the up-regulation of MAPK signaling pathway. ConclusionThe sesquiterpenoid M36 isolated from Myrrha inhibits the proliferation of human hepatoma HepG2 cells and promotes apoptosis and autophagy， which may be related to the activation of the MAPK signaling pathway.

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.

Wenyang Jieyu Prescription Regulates Hippocampal Neuron Apoptosis and Improves Synaptic Plasticity in Depressed Mice via BDNF/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomly assigned into a control group （n=10） and a modeling group （n=50）. Maternal separation combined with restraint stress was adopted to establish the mouse model of depression， and the modeled mice were randomized into model， Wenyang prescription， Jieyu prescription， Wenyang Jieyu prescription， and fluoxetine groups （n=10） on the weaning day （PD21）. From PD21 to PD111， the mice were fed with the diets mixed with corresponding medicines. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then conducted to evaluate the depression， memory， and learning abilities of mice. Immunohistochemistry （IHC） was employed to measure the atomic absorbance （AA） of postsynaptic density protein 95 （PSD95） in the hippocampus. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of hippocampal neurons. Western blot was employed to determine the protein levels of brain-derived neurotrophic factor （BDNF）， phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B （p-TrkB/TrkB）， phosphorylated protein kinase B/protein kinase B （p-Akt/Akt）， phosphorylated mammalian target of rapamycin/mammalian target of rapamycin （p-mTOR/mTOR）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cysteinyl aspartate-specific proteinase-3 （Caspase-3）， synaptophysin （Syn）， and PSD95. ResultCompared with the control group， the modeling decreased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.01）. Furthermore， it decreased the expression of PSD95， increased the neuron apoptosis in the hippocampus （P<0.01）， down-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and up-regulated the protein levels of Bax and Caspase-3 （P<0.05） in the hippocampus. Compared with the model group， Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. Moreover， the drugs increased the expression of PSD95， reduced the neuron apoptosis （P<0.01）， up-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and down-regulated the protein levels of Bax and Caspase-3 （P<0.01）. ConclusionWenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice. It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.

Inhibition of Oxidative Stress of Sciatic Nerve in Diabetic Rats by Tangbikang Granules Regulating AMPK/PGC-1α/SIRT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Tangbikang granules on oxidative stress of sciatic nerve in diabetic rats by regulating adenylate activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α/mitochondrial Sirtuins 3 （AMPK/PGC-1α/SIRT3） signaling pathway. MethodThe spontaneous obesity type 2 diabetes model was established using ZDF rats. After modeling， they were randomly divided into high， medium， and low dose Tangbikang granule groups （2.5， 1.25， 0.625 g·kg-1·d-1） and lipoic acid group （0.026 8 g·kg-1·d-1）， and the normal group was set up. The rats were administered continuously for 12 weeks after modeling. The blood glucose of rats was detected before intervention and at 4， 8， 12 weeks after intervention. At the 12th week， motor nerve conduction velocity （MNCV）， sensory nerve conduction velocity （SNCV）， nerve blood flow velocity， mechanical pain threshold， and thermal pain threshold were detected. The sciatic nerve was taken for hematoxylin-eosin （HE） staining to observe the tissue morphology. The ultrastructure of the sciatic nerve was observed by transmission electron microscope. The expression levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） in sciatic nerve were determined by enzyme-related immunosorbent assay （ELISA）. The mRNA expressions of AMPKα， AMPKβ， PGC-1α， and SIRT3 in sciatic nerve were determined by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the normal group， fasting blood glucose in the model group was increased at each time point （P<0.01）. The mechanical pain threshold was decreased （P<0.05）， and the incubation time of the hot plate was extended （P<0.01）. MNCV， SNCV， and nerve blood flow velocity decreased （P<0.05）. The expression level of SOD was decreased （P<0.01）. The expression levels of MDA， IL-1β， and TNF-α were increased （P<0.01）. The mRNA expression levels of AMPKα， AMPKβ， PGC-1α， and SIRT3 were decreased （P<0.01）. The structure of sciatic nerve fibers in the model group was loose， and the arrangement was disordered. The demyelination change was obvious. Compared with the model group， the fasting blood glucose of rats in the high dose Tangbikang granule group was decreased after the intervention of eight weeks and 12 weeks （P<0.01）. The mechanical pain threshold increased （P<0.05）. The incubation time of the hot plate was shortened （P<0.01）. MNCV， SNCV， and Flux increased （P<0.05）. The expression level of SOD was increased （P<0.01）. The expression levels of MDA， IL-1β， and TNF-α were decreased （P<0.01）. The mRNA expression levels of AMPKα， AMPKβ， PGC-1α， and SIRT3 were increased （P<0.01）. The sciatic nerve fibers in the high-dose Tangbikang granule group were tighter and more neatly arranged， with only a few demyelinating changes. The high， medium， and low dose Tangbikang granule groups showed a significant dose-effect trend. ConclusionTangbikang granules may improve sciatic nerve function in diabetic rats by regulating AMPK/PGC-1α/SIRT3 signaling pathway partly to inhibit oxidative stress.

Exploring Effect and Mechanism of Astragaloside on Repair and Healing in Chronic Non-healing Wound Rat Model Based on SIRT1 and PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the promotional effect of astragaloside on the repair and healing of chronic non-healing wounds and its mechanism. MethodA total of 60 male SD rats were constructed with full-layer skin defect wounds on the back， and except for the control （Con） group， the rest were constructed with non-healing wounds， which were then randomly divided into the sham-operation （sham） group， the low-dose astragaloside group， the high-dose astragaloside group， the astragaloside + LY294002 ［phosphatidylinositol 3-kinase （PI3K） inhibitor］ group， and the astragaloside + EX527 ［silencing regulatory protein 1 （SIRT1） inhibitor］ group. The percentage of wound area in each group was observed on the 2nd， 4th， 6th， and 8th days after wound molding. Collagen type Ⅰ alpha 1 （COL1A1） and alpha smooth muscle actin （α-SMA） expressions in the wound tissue were detected by immunofluorescence. Hematoxylin and eosin （HE） staining was performed to determine the pathological structure of the wound. The mRNA expression of inflammatory factors in the wound was measured by real-time polymerase chain reaction （Real-time PCR）， and the expression of proteins related to the SIRT1/ nuclear factor （NF）-κB and PI3K/protein kinase B （Akt） signaling pathways in the wound was tested by Western blot. ResultCompared with the sham group， the percentage of postoperative wound area of rats in both low-dose and high-dose astragaloside groups gradually decreased with time， and the efficacy of the high-dose astragaloside group was better. Compared with the Con group， the fluorescence intensity of COL1A1 in wound tissue of the sham group decreased， while the expression of α-SMA increased. The epithelial tissue was severely damaged， with an increase in the thickness， and a large number of inflammatory cells were seen in the infiltration. The mRNA expression of tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and inducible nitric oxide synthase （iNOS） was elevated. The protein expression of NF-κB p65， p-PI3K/PI3K， and p-Akt/Akt was elevated， while SIRT1 expression was decreased （P<0.05）. Compared with the sham group， the fluorescence intensity of COL1A1 and α-SMA increased after astragaloside treatment. The number of epithelial cells increased， and the thickness decreased. The inflammatory cells decreased， and the amount of collagen increased. The mRNA expression of TNF-α， IL-1β， IL-6， and iNOS was decreased， and the protein expression of NF-κB p65， p-PI3K/PI3K， and p-Akt/Akt was decreased. SIRT1 was elevated， and the effect was better in the high-dose astragaloside group （P<0.05）. Compared with the high-dose astragaloside group， inhibition of the PI3K/Akt and SIRT1 pathways by LY294002 and EX527 prevented the therapeutic efficacy of astragaloside on chronic non-healing wounds. ConclusionThe topical application of astragaloside significantly promotes the healing of chronic non-healing wounds in rats， and the mechanism may be related to the activation of the PI3K/Akt pathway and the SIRT1/NF-κB pathway.

Treatment of Cerebral Ischemia-reperfusion Injury by Traditional Chinese Medicine Based on PI3K/Akt Signaling Pathway： A Review / 中国实验方剂学杂志

Cerebral ischemia/reperfusion injury （CIRI） is a complex cascade reaction process in which the blood flow and oxygen supply of brain tissue in the infarcted area recover after cerebral ischemia， resulting in secondary injury of ischemic brain tissue. At present， thrombolysis as soon as possible and restoration of cerebral blood supply are still the only strategies for the treatment of stroke， but a considerable number of patients' symptoms will be more serious after reperfusion， making patients face adverse outcomes such as neurological function injury and even death and seriously affecting the quality of life and safety of patients. Therefore， an in-depth exploration of the mechanism and treatment strategy of CIRI has important clinical significance. The phosphatidylinositol 3- kinase （PI3K）/protein kinase B （Akt） signaling pathway is one of the classic anti-apoptosis/reproductive-promoting signal transduction pathways， which is responsible for cell proliferation， growth， and differentiation. It is the key cascade signaling pathway of CIRI， located at the core site in many mechanisms such as mitochondrial disorder， apoptosis， autophagy， oxidative stress， and inflammation. It is closely related to the occurrence and development of CIRI. Traditional Chinese medicine has been used in the clinical treatment of stroke and its complications for thousands of years， and the clinical effect of traditional Chinese medicine in the prevention and treatment of CIRI has been affirmed by a large number of research results in recent years. It is further clarified that the monomers， active components， and their compound prescriptions of traditional Chinese medicine can directly or indirectly regulate the PI3K/Akt signaling pathway by virtue of the biological advantages of multi-targets， multi-components， and multi-pathways and play an overall protective role in CIRI. By analyzing the related research progress of traditional Chinese medicine in China and abroad in recent years， the authors summarized the role and mechanism of regulating the PI3K/Akt signaling pathway in the prevention and treatment of CIRI， so as to provide further theoretical basis for the study of the mechanism of clinical prevention and treatment of CIRI.

Huaiqihuang Granules Affect Differentiation of Th17 Cells in IgA Vasculitis Nephritis Mice by Regulating AMPK/ACC Pathway / 中国实验方剂学杂志

ObjectiveTo observe the intervention effect of Huaiqihuang granules （HQH） on immunoglobulin A vasculitis nephritis （IgAVN） mice and explore the underlying therapeutic mechanism. MethodFifty SPF-grade male Kunming mice were randomly divided into a normal group， an IgAVN model group， a dexamethasone group （2.5 mg·kg-1·d-1）， a low-dose HQH group （4 g·kg-1·d-1）， and a high-dose HQH group （8 g·kg-1·d-1）. The mouse model was established using oral administration of gliadin combined with intravenous injection of India ink. After successful modeling， the mice were euthanized after 4 weeks of gastric gavage according to groups. The 24 h urinary total protein （24 h UTP）， urine β2-microglobulin （β2-MG）， serum total protein， albumin， IgA， etc. were detected in each group. Flow cytometry was used to determine the proportion of T helper 17 （Th17） cells in spleen cell suspension. Western blot was employed to detect the expression of adenosine 5'-monophosphate-activated protein kinase α （AMPKα）， phosphorylated AMPKα （p-AMPKα）， acetyl-CoA carboxylase 1 （ACC1）， and phosphorylated ACC1 （p-ACC1） in Th17 cells. Pathological changes in the spleen and kidneys were observed. ResultCompared with the normal group， the IgAVN model group showed significant increases in 24 h UTP， urine β2-MG， total cholesterol （P<0.05）， serum interleukin-17 （IL-17）， IgA， Th17 proportion in the spleen cell suspension， and IL-17 expression in the spleen tissue （P<0.01）， and significantly decreased serum total protein， albumin， p-AMPKα/AMPKα， and p-ACC1/ACC1 expression of Th17 cells （P<0.01）. Compared with the IgAVN model group， in the 4th week， the 24 h UTP， urine β2-MG， serum IL-17， IgA levels， and renal IgA deposition were significantly reduced in each treatment group （P<0.01）， and the Th17 proportion and IL-17 expression in spleen tissue were significantly decreased （P<0.05， P<0.01）. Serum albumin levels significantly increased （P<0.05）. Compared with the IgAVN model group， the dexamethasone group and the high-dose HQH group showed increases in serum total protein （P<0.01）， p-AMPKα/AMPKα， and p-ACC1/ACC1 expression of Th17 cells （P<0.05， P<0.01）. The high-dose HQH group showed a significant decrease in total cholesterol level （P<0.05）. Various treatment groups showed different degrees of improvement in spleen and kidney pathological changes. ConclusionHQH may affect Th17 cell differentiation by regulating the AMPK/ACC pathway， correcting immune inflammatory disorders， and exerting therapeutic effects on IgAVN.

Mechanism of Modified Tianwang Buxindan on Skin of Sleep-deprived Mice Through PI3K/Akt/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Tianwang Buxindan （MTBD） on the skin of sleep-deprived （SD） mice and investigate its mechanism. MethodSixty 2-month-old female Kunming mice were randomly divided into a blank group， a model group， a vitamin C （VC， 0.08 g·kg-1）， and MTBD low-， medium-， and high-dose groups （6.5， 12.5， 25 g·kg-1）. Except for the blank group， the other groups were subjected to SD mouse model induction （using multiple platform water environment method for 18 hours of sleep deprivation daily from 15：00 to next day 9：00）， continuously for 14 days， and caffeine （CAF， 7.5 mg·kg-1） was injected intraperitoneally from the 2nd week onwards， continuously for 7 days. While modeling， the blank group and the model group were administered with normal saline （0.01 mL·g-1）， and the other groups received corresponding drugs for treatment. On the day of the experiment， general observations were recorded （such as body weight， spirit， fur， and skin）. After sampling， skin tissue pathological changes were observed under an optical microscope using hematoxylin-eosin （HE） and Masson staining methods. Skin thickness and skin moisture content were measured. Biochemical assay kits were used to detect skin hydroxyproline （HYP） content， skin and serum superoxide dismutase （SOD） activity， and malondialdehyde （MDA） content. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum interleukin （IL）-6， tumor necrosis factor （TNF）-α， and IL-1β levels in mice. Western blot was used to detect skin tissue type Ⅰ collagen （ColⅠ）， type Ⅲ collagen （ColⅢ）， phosphatidylinositol 3-kinase （PI3K）， phosphorylated （p）-PI3K， protein kinase B （Akt）， p-Akt， nuclear factor E2-related factor 2 （Nrf2）， heme oxygenase （HO）-1， and nuclear factor （NF）-κB protein expression. ResultCompared with the blank group， the model group showed varying degrees of changes. In general， signs of aging such as reduced body weight （P<0.01）， listlessness， dull fur color， and formation of wrinkles on the skin appeared. Tissue specimen testing revealed skin thinning， flattening of the dermoepidermal junction （DEJ）， and reduced collagen fibers under the optical microscope. Skin thickness and moisture content decreased， skin tissue HYP content significantly decreased （P<0.01）， skin and serum SOD activity significantly decreased （P<0.01）， and MDA content significantly increased （P<0.01）. Serum IL-6， TNF-α， and IL-1β levels significantly increased （P<0.01）. Skin ColⅠ， ColⅢ， p-PI3K/PI3K， p-Akt/Akt， Nrf2， and HO-1 protein expression significantly decreased （P<0.05， P<0.01）， and NF-κB expression increased （P<0.01）. Compared with the model group， the VC group and the MTBD low-dose group showed increased skin moisture content， HYP content， SOD activity， and ColⅠ， ColⅢ， p-PI3K/PI3K protein expression （P<0.05， P<0.01）， and decreased serum MDA content （P<0.05）. In addition， a decrease in serum IL-6 and IL-1β levels was detected in the MTBD low-dose group （P<0.05）， while the above indicators in the MTBD medium- and high-dose groups improved （P<0.05， P<0.01）. ConclusionSleep deprivation accelerates the aging process of the skin in SD model mice. MTBD can improve this phenomenon， exerting anti-inflammatory and antioxidant effects， and its mechanism of action may be related to the activation of the PI3K/Akt/Nrf2 signaling pathway.

Mechanism of Xiezhuo Jiedu Recipe in Preventing Colorectal Adenoma in Mice Through miRNA-34a-5p/PI3K/Akt Signalling Pathway / 中国实验方剂学杂志

ObjectiveKey microRNAs （miRNAs） of colorectal adenoma （CRA） were identified and analyzed by bioinformatics methods， and differentially expressed genes （DEGs） were screened to construct regulatory relationships. The mechanism of Xiezhuo Jiedu recipe in preventing CRA was speculated and verified by animal experiments. MethodThe miRNAs dataset GSE50194 was obtained from the Gene Expression Omnibus （GEO） database of intestinal mucosal tissue of CRA patients， and the differentially expressed miRNAs were screened by GEO2R and Excel. TargetScan， miRTarbase， and miRDB databases were used to predict the target genes of the differentially expressed miRNAs， and an intersection was obtained. Key DEGs were screened through the STRING database and Cytoscape software， and the TRRUST database was used to predict downstream binding transcription factors （TFs）. The mRNA intersection was enriched by gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） in the Metascape database. DIANA TOOLS were applied to perform KEGG enrichment analysis of key miRNAs， and the key signaling pathways were selected for animal experiments. In animal experiments， the CRA mice model was established by using sodium glycan sulfate （DSS） drinking combined with intraperitoneal injection of azomethane oxide （AOM）， and Xiezhuo Jiedu recipe and aspirin were given by intragastric administration at the same time. The experiment lasted for nine weeks. The pathological changes in intestinal tissue were observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of miR-34a-5p in adenoma tissue. Protein expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphoryl-PI3K （p-PI3K）， phosphoryl-Akt （p-Akt）， and B cell lymphoma （Bcl）-2 were detected by Western blot. The expression of Cyclin D1 （CCND1） was detected by immunohistochemistry （IHC）. In situ terminal transferase labeling （TUNEL） was used to detect apoptosis of adenoma tissue cells. ResultThe GEO database screened the GSE50194 dataset， and miR-34a-5p was selected as the research object from CRA and normal tissue. A total of 93 DEGs were selected. Among them， GO and KEGG enrichment analyses were closely related to biological processes such as transcriptional regulatory complex， RNA polymerase Ⅱ transcriptional regulatory complex， enzyme-linked receptor protein signaling pathway， and DNA-binding transcriptional activator activity， cancer pathway， PI3K/Akt pathway， etc. miR-34a-5p is mainly enriched in PI3K/Akt， cell cycle， and colorectal cancer pathways. Five key DEGs were screened out through the Matescape database， among which Bcl-2 and CCND1 were the key DEGs of miR-34a-5p. Further screening of the TFs of key DEGs revealed that E2F transcription factor 1 （E2F1） and tumor protein P53 （TP53） were the main TFs of Bcl-2 and CCND1. Animal experiments showed that Xiezhuo Jiedu recipe could effectively up-regulate mRNA level of miR-34a-5p， down-regulate the expression of PI3K， Akt， Bcl-2， p-PI3K， and p-Akt proteins in the intestinal tissue of CRA mice， down-regulate the positive expression rate of CCND1， and increase the apoptosis rate of intestinal epithelial cells. ConclusionIt is speculated that Xiezhuo Jiedu recipe may inhibit the abnormal proliferation and promote the apoptosis of intestinal epithelial cells in CRA mice by regulating the miR-34a-5p/PI3K/Akt signaling pathway， thus playing a role in the prevention of CRA.

Treatment of Exercise-induced Fatigue with Buzhong Yiqitang Through Adipor1/AMPK/PGC-1α to Regualte Lipid Metabolism / 中国实验方剂学杂志

ObjectiveTo discuss the impact of Buzhong Yiqitang on lipid metabolism in skeletal muscle of exercise-induced fatigue （EIF） mice through adiponectin receptor 1 （Adipor1）/adenosine 5'-monophosphate（AMP）-activated protein kinase （AMPK）/peroxisome proliferator-activated receptor gamma coactivator-1α （PGC-1α）. MethodC57BL6J mice were randomly divided into the control group， model group， low， middle， and high dose groups of Buzhong Yiqitang， and vitamin C group. No intervention was given to the control group， while the other groups were subjected to exhaustive swimming training to establish the EIF model. One hour before exhaustion， 0.2 mL distilled water was given to the control group and the model group， while the mice in the low， middle， and high dose groups of Buzhong Yiqitang were given intragastrically Buzhong Yiqitang of 4.1， 8.2， and 16.4 g·kg-1， respectively， and the vitamin C group was given vitamin C of 0.04 g·kg-1 via gavage for a duration of six weeks. After six weeks of the experiment， the growth rate of body weight， organ index， and exhaustive swimming time were calculated. Enzyme colorimetry was utilized to detect the levels of blood urea nitrogen （BUN）， creatine kinase acid （CK）， lactate dehydrogenase acid （LDH）， and lactic acid （LD）. The pathological changes of skeletal muscle were observed using hematoxylin -eosin （HE） staining， while the ultrastructure of skeletal muscle was observed with transmission electron microscope （TEM）. The contents of free fatty acids （NEFA） and triglyceride acid （TG） in serum were also examined by microplate method. The protein expressions of Adipor1， p-AMPK/AMPK， PGC-1α， and HK2 in the skeletal muscle were measured by Western blot. ResultCompared with those of the control group， the growth rate of body weight and thymus index of the model group were decreased， and the serum levels of BUN， CK， LD， and LDH were increased （P<0.01）. The contents of NEFA and TG were decreased （P<0.01）， and the protein expression of Adipor1， p-AMPK/AMPK， PGC-1 α， and HK2 in the skeletal muscle decreased （P<0.05， P<0.01）. Compared with those in the model group， the growth rate of body weight， thymus index， and exhaustive swimming time were significantly increased （P<0.01）， and the levels of BUN， CK， LD， and LDH dropped in the high dose group of Buzhong Yiqitang （P<0.01）. The levels of NEFA and TG were greatly improved （P<0.01）. The protein expressions of Adipor1， p-AMPK/AMPK， PGC-1α， and HK2 in the skeletal muscle were significantly increased （P<0.05， P<0.01）. Compared with those in the model group， the thymus index and exhaustive swimming time were significantly increased in the vitamin C group， and the levels of BUN， CK， and LD dropped （P<0.05， P<0.01）. The levels of NEFA and TG were improved significantly （P<0.01）， and the protein expression of Adipor1 in skeletal muscle was increased greatly （P<0.01）. ConclusionBuzhong Yiqitang can delay the development of EIF， which may be connected with the regulation of the Adipor1/AMPK/PGC-1α signaling pathway and the improvement of the utilization rate of skeletal muscle to fat.

Influence of Didang Xianxiong Decoction on Apoptosis of Podocytes in Diabetes Rats Through PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the protective effect of Didang Xianxiong decoction on the kidneys of diabetic rats， its regulation on the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， and its influence on podocyte apoptosis and explore the mechanism of Didang Xianxiong decoction in improving diabetic nephropathy. MethodThe diabetic model was established by a single intraperitoneal injection of streptozotocin （STZ） solution of 55 mg·kg-1. The successfully replicated model rats were randomly divided into the model group， Didang Xianxiong decoction group （8.10 g·kg-1）， Xiao Xianxiongtang group （4.05 g·kg-1）， Didangtang group （4.05 g·kg-1）， and alagebrium （ALT-711） group （3 mg·kg-1）， with six rats in each group. In addition， six rats were included in the blank group. After continuous administration for eight weeks， hematoxylin-eosin （HE） staining was used to observe the pathological changes in rats' kidney tissue. Masson staining was used to observe the degree of collagen deposition. Periodic acid-Schiff （PAS） staining was used to observe basement membrane lesions， and immunohistochemistry was used to detect the expression of phosphorylation （p）-PI3K and p-Akt proteins in rats' kidney tissue. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） method was used to detect podocyte apoptosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of PI3K and Akt in rats' kidney tissue. Western blot was used to detect the protein expression of PI3K， p-PI3K， Akt， p-Akt， B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， phosphorylation glycogen synthase kinase-3β （p-GSK-3β）， and Caspase-3 in the kidney tissue. ResultCompared with the normal group， the model group had compensatory expansion of glomeruli， proliferation of mesangial cells， a large amount of collagen deposition in the mesangial stroma， thickening of the basement membrane， decreased mRNA expression of PI3K and Akt， and inhibition of PI3K and Akt protein phosphorylation （P<0.01）. It also underwent enhanced apoptotic signaling， decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）， and increased expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）. Compared with the model group， Didang Xianxiong decoction significantly improved kidney tissue pathology， increased mRNA expression of PI3K and Akt （P<0.01）， significantly up-regulated phosphorylation levels of PI3K and Akt proteins （P<0.01） and Bcl-2 expression （P<0.01）， downregulated the expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）， and weakened podocyte apoptotic signaling. ConclusionDidang Xianxiong decoction may promote the activation of the PI3K/Akt signaling pathway， inhibit podocyte apoptosis， and thus slow down the progression of diabetic nephropathy.

Inhibition of Alcoholic Liver Injury by Paeonol Through Eubacterium-mediated TGR5/PKA/CREB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate whether paeonol exerts a protective effect on mice with alcoholic liver injury by regulating the takeda G-protein-coupled receptor 5 （TGR5）/protein kinase A （PKA）/cAMP response binding element （CREB） signaling pathway mediated by Eubacterium. MethodC57BL/6 mice were randomly divided into five groups： normal group， model group， paeonol group （480 mg·kg-1）， antibiotic group （Abs group）， and antibiotic + paeonol group. Lieber-DeCarli liquid was used to feed C57BL/6 mice on the second day of modeling for 10 days. The blood lipids， liver function， inflammatory factors， and oxidative stress levels in mice were measured. Hematoxylin-eosin staining （HE） and oil red O staining were used to observe the morphological changes and fat accumulation in liver tissue. 16S rDNA sequencing was used to detect the diversity of intestinal microbiota in the blank， model， and paeanol groups. Western blot was used to detect the effect of paeonol on the expression levels of protein related to the signaling pathway of atresia band protein 1 （ZO-1）， Claudin-1， and TGR5/PKA/CREB in mouse ileal tissue. ResultCompared with those in the blank group， the blood lipids， liver function， oxidative stress levels， and the expression of inflammatory factors in the model group increased （P<0.01）， and the liver fat vacuoles were obvious. The ileal mucosa was seriously damaged， and the protein contents of ZO-1， Claudin-1， and TGR5/PKA/CREB in the ileal tissue decreased significantly （P<0.01）. The intestinal microbiota changed， and the proteobacteria phylum increased significantly. The ratio of Bacteroidetes to Firmicutes decreased. The relative abundance of Dubosiella newyorkensis， Lactobacillus， Bifidobacterium， and other genera decreased， while the relative abundance of Escherichia-Shigella， Morganella， Providencia， and Proteus increased significantly. Compared with the model group， paeonol significantly reduced the blood lipids， liver function， oxidative stress levels， and expression of inflammatory factors in mice with alcohol diet-induced liver injury （P<0.05）， decreased liver fat vacuoles， improved and restored the ileal intestinal barrier， and restored the normal structure of hepatocytes and ileal cells. The intestinal microbiota disorder caused by alcohol was improved， and the relative abundance of beneficial bacteria such as Eubacterium spp. was increased. The protein expression levels of ZO-1， Claudin-1， and TGR5/PKA/CREB in ileal tissue were increased （P<0.05）. ConclusionPaeonol has a protective effect on alcoholic liver injury in mice， and the mechanism of action is achieved by regulating the Eubacterium-mediated TGR5/PKA/CREB signaling pathway to ensure anti-inflammatory effect and improve the intestinal barrier.

Mechanism of malignant behavior in cervical cancer cells based on NOD2-mediated AMPK/mTOR signaling pathway / 安徽医科大学学报

Objective To explore the mechanism of malignant behavior of cervical cancer(CC)cells based on AMP-activated protein kinase(AMPK)/rapamycin target protein(mTOR)signaling pathway mediated by nucleo-tide-binding oligomerization domain receptor 2(NOD2).Methods Bioinformatics analysis was performed to deter-mine the expression of NOD2 in CC tissue.Plasmids targeting NOD2(shNOD2)and shRNAs negative control(shNC),NOD2 overexpression(NOD2)and vectors(Vec)were transfected into CC cells.The effect of NOD2 on the growth of CC cells was determined by cell counting kit-8 assay,colony formation and Transwell cell invasion as-say.Transcriptome analysis was performed by high throughput RNA sequencing(RNA-Seq).Western blot was used to detect the expression of NOD2,AMPK/mTOR signaling pathway and autophagy protein in the cell line.24 female BALB/c nude mice were randomly divided into four groups,with 6 mice in each group:vector group(Vec group),NOD2 overexpression group(NOD2 group),shNC group and shNOD2 group.The distant metastasis mod-el was established in mice,and the fluorescence intensity of lung metastasis was monitored and the number of lung metastasis nodules was counted.Results On-line database analysis showed that the expression of NOD2 in CC tis-sues was significantly higher than that in normal tissues,and there were significant differences in the mRNA expres-sion of NOD2 in different stages of CC(P＜0.05).In addition,the high expression of NOD2 was associated with poor overall survival and disease-free survival(P＜0.05).NOD2 overexpression promoted the proliferation,colony formation,migration and invasion of CC cells,while NOD2 knock-down was the opposite.Consistent with the re-sults in vitro,the lung colonization and lung metastasis of CC cells in NOD2 group were significantly higher than those in Vec group(P＜0.05),while those in shNOD2 group were significantly lower than those in shNC group(P＜0.05).RNA-Seq results showed that the expression of NOD2 was significantly related to AMPK signal activa-tion,mTOR signal inhibition,autophagy regulation pathway activation and autophagy formation.Compared with shNC group,the expression of phosphorylated AMPK and LC3 protein decreased significantly in shNOD2 group(P＜0.05),and the expression levels of phosphorylated mTOR and p62 protein increased significantly(P＜0.05).Compared with Vec group,the expression levels of LC3 and AMPK protein in NOD2 group increased significantly(P＜0.05),and the expression levels of phosphorylated mTOR and p62 protein decreased significantly(P＜0.05).Compared with shNC group,the point accumulation of GFP-mRFP-LC3 in shNOD2 group decreased signifi-cantly(P＜0.05).Compared with Vec group,the point accumulation of GFP-mRFP-LC3 increased significantly(P＜0.05).Conclusion NOD2 may promote the proliferation,migration and invasion of CC through AMPK/mTOR signal,and its mechanism partly involves autophagy activation.

Improvement effect of ligustilide on rats with heart failure by regulating PKD1/HIF-1α/VEGF pathway / 吉林大学学报(医学版)

Objective:To discuss the effect of ligustilide on the cardiac function and angiogenesis in the rats with heart failure,and to clarify its regulatory effect on protein kinase D1(PKD1)/hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)pathway.Methods:The SD rats were randomly divided into sham operation group,model group,ligustilide group,PKD1/HIF-1α/VEGF signaling pathway inhibitor CID755673(CID)group,and ligustilide+CID group.The heart failure rat model was established by ligation of the left anterior descending coronary artery.The rats in ligustilide group were injected intravenously with 20 mg·kg-1 ligustilide,the rats in CID group were injected intraperitoneally with 50 mg·kg-1 CID,and the rats in ligustilide+CID group were injected intraperitoneally with 50 mg·kg-1 CID followed by intravenous injection of 20 mg·kg-1 ligustilide,once per day for 4 consecutive weeks.The cardiac function indexes of the rats in various groups were detected by echocardiography;the percentages of myocardial infarction areas of the rats in various groups were detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining;the pathomorphology of myocardium tissue of the rats in various groups was observed by HE staining;the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in ischemic area of myocardium tissue of the rats in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.Results:Compared with sham operation group,the rats in model group and CID group had altered myocardial cell morphology,increased intercellular gaps,disorganized arrangement,visible muscle fiber breaks and inflammatory cell infiltration;the rats in ligustilide group and ligustilide+CID group had relatively orderly myocardial fiber arrangement,fewer myocardial fiber breaks and decreased number of inflammatory cells.Compared with sham operation group,the left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)of the rats in model group were decreased(P<0.05),the left ventricular end-systolic diameter(LVESD)and left ventricular end-diastolic diameter(LVEDD)were increased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were decreased(P<0.05).Compared with model group,the LVEF and LVFS of the rats in ligustilide group were increased(P<0.05),the LVESD and LVEDD were decreased(P<0.05),the percentage of myocardium infarction area was decreased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were increased(P<0.05);compared with model group,the LVEF and LVFS of the rats in CID group were decreased(P<0.05),the LVESD and LVEDD were increased(P<0.05),the percentage of myocardium infarction area was increased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were decreased(P<0.05);compared with ligustilide group,the LVEF and LVFS of the rats in ligustilide+CID group were decreased(P<0.05),the LVESD and LVEDD were increased(P<0.05),the percentage of myocardium infarction area was increased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were decreased(P<0.05);compared with CID group,the LVEF and LVFS of the rats in ligustilide+CID group were increased(P<0.05),the LVESD and LVEDD were decreased(P<0.05),the percentage of myocardium infarction area was decreased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were increased(P<0.05).Conclusion:Ligustilide can promote the angiogenesis,reduce the myocardium infarction area,and improve the cardiac function in the rats with heart failure;it works through activation of the PKD1/HIF-1α/VEGF pathway.

Effect of downregulating proline-rich protein 11 expression on drug resistance of esophageal cancer drug resistant cell EC9706/DDP and its mechanism / 吉林大学学报(医学版)

Objective:To discuss the effect of downregulating the proline-rich protein 11(PRR11)expression on drug resistance of the esophageal cancer drug resistant cells,and to clarify the related mechanism.Methods:The drug resistant cells EC9706/cisplatin(DDP)were established by incrementally stimulating the human esophageal cancer EC9706 cells with the increasing concentrations of DDP.The drug sensitivity of the EC9706/DDP cells was detected by MTT assay;the expression levels of PRR11 mRNA and protein in the EC9706/DDP cells and their parent EC9706 cells were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.The EC9706/DDP cells were divided into control group,sh-NC group(infected with sh-NC),sh-PRR11 group(infected with sh-PRR11),sh-NC+DDP group(infected with sh-NC and treated with 4 mg·L-1 DDP),and sh-PRR11+DDP group(infected with sh-PRR11 and treated with 4 mg·L-1 DDP).The expression levels of PRR11 mRNA in the cells in various groups were detected by RT-qPCR method;the expression levels of PRR11,phosphoinositide 3-kinase(PI3K)p110α,protein kinase B(AKT),phosphorylated AKT(p-AKT),P-glycoprotein(P-gp),and multidrug resistance-associated protein 1(MRP1)proteins in the cells in various groups were detected by Western blotting method;the apoptotic rates of the cells in various groups were detected by flow cytometry.Results:The DDP-resistant cell line EC9706/DDP was successfully obtained,and the drug resistance index was 7.23±0.86.Compared with the EC9706 cells,the expression levels of PRR11 mRNA and protein in the EC9706/DDP cells were increased(P<0.05).Compared with control and sh-NC groups,the expression levels of PRR11 mRNA and protein in the cells in sh-PRR11 group were decreased(P<0.05),and the 50%inhibitory concentration(IC50)of DDP was decreased(P<0.05).Compared with sh-NC group,the expression levels of PI3K p110α,p-AKT,P-gp,and MRP1 proteins in the cells in sh-NC+DDP and sh-PRR11 groups were decreased(P<0.05),and the apoptotic rate of the cells was increased(P<0.05).Compared with sh-NC+DDP group and sh-PRR11 group,the expression levels of PI3K p110α,p-AKT,P-gp,and MRP1 proteins in the cells in sh-PRR11+ DDP group were increased(P<0.05),and the apoptotic rate of the cells was increased(P<0.05).Conclusion:Downregulating the expression of PRR11 gene in the drug resistant EC9706/DDP cells can inhibit the expressions of drug resistance-related proteins,reverse the resistance to DDP,and induce the apoptosis;its mechanism may be related to the inhibition of activation of the PI3K/AKT signaling pathway.

Inhibitory effect of downregulating HMGB2 expression on epithelial-mesenchymal transition of liver cancer LM3 cells and its AKT/m TOR signaling pathway mechanism / 吉林大学学报(医学版)

Objective:To discuss the effect of downregulating of high mobility group box protein 2(HMGB2)expression on the biological behavior of the liver cancer cells and the epithelial-mesenchymal transition(EMT)process,and to clarify its mechanism.Methods:The human liver cancer LM3 cells at logarithmic growth phase were divided into negative control group and HMGB2 RNA interference group(HMGB2 siRNA group);the cells in two groups were transfected with RNA oligonucleotides(RNA oligos)with irrelevant sequences and RNA oligos designed to knock down HMGB2,and the Lipofectamine 2000 was regarded as the vector.The expression levels of HMGB2 mRNA and protein in the cells in two groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods;cell scratch assay and Transwell chamber assay were used to detect the migration and invasion abilities of the cells in two groups;the expression levels of E-cadherin,N-cadherin,and Vimentin proteins and protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway related proteins in the cells in two groups were detected by Western blotting method.Results:Compared with negative control group,the expression levels of HMGB2 mRNA and protein in the cells in HMGB2 siRNA group were significantly decreased(P<0.05),the cell scratch healing rate was significantly decreased(P<0.01),the number of invasion cells was significantly decreased(P<0.01),and the expression level of E-cadherin protein in the cells was significantly increased(P<0.01),while the expression levels of N-cadherin,Vimentin,mTOR,AKT,and phosphorylated AKT(p-AKT)proteins in the cells were significantly decreased(P<0.05 or P<0.01).Conclusion:Downregulating the expression of HMGB2 can reduce the migration and invasion abilities of the liver cancer LM3 cells and inhibit the EMT,and its mechanism may be related to regulating the expression of the AKT/mTOR pathway related proteins.

Expression and significance of PIK3CA,p-AKT and PTEN in sinonasal squamous cell carcinoma / 重庆医学

Objective To investigate the expression of PIK3CA,phosphorylated protein kinase B(p-AKT)and phosphatase and tensin homologue deleted on chromosome 10(PTEN)in sinonasal squamous cell carcinoma(SNSCC).Methods The expressions of PIK3CA and PTEN in head and neck squamous cell carci-noma(HNSCC)were analyzed through the data set of HNSCC in the cancer genome map of UCSC Xena data-base.The immunohistochemical SP method was used to measure the expression of PIK3CA,p-AKT and PTEN in 43 cases of SNSCC tissues,20 cases of normal inferior concha tissues.The relationship between the expressions of PIK3CA,p-AKT and PTEN protein with the clinicopathological features and prognosis of the patients with SNSCC was analyzed.Results The results of bioinformatic analysis showed that PIK3CA mR-NA expression in HNSCC tissues was higher than that in paracancerous tissues(P＜0.01),while the PTEN mRNA expression was lower than that in paracancerous tissues(P＜0.05).The immunohistochemical detec-tion results showed that the positive expressions rates of PIK3CA and p-AKT proteins in normal nasal mucosa tissues were significantly lower than those in SNSCC tissues,while the positive expression rate of PTEN pro-tein in SNSCC tissues was significantly higher than that in normal inferior nasal concha mucosa tissues,and the differences were statistically significant(P＜0.01).The expressions of PIK3CA and p-AKT protein were related to the clinical stage,differentiation degree and primary site(P＜0.05),but were not related to age,gender,smoking and drinking(P＞0.05);the PTEN protein expression was not related with the clinical stage,differentiation degree,primary site,age,smoking and drinking(P＞0.05).The Spearman analysis showed that the expression of PIK3CA in SNSCC tissues was positively correlated with p-AKT protein ex-pression(r=0.664,P＜0.01),and PIK3CA was negatively correlated with PTEN protein(r=-0.414,P＜0.01).The expression of p-AKT was negatively correlated with PTEN protein(r=-0.453,P＜0.01).The Kaplan-Meier analysis showed that the median survival time of the patients with PIK3CA and p-AKT protein positive expression was shorter than that of the patients with negative expression(P＜0.01).There was no statistically significant difference in median survival between the patients with PTEN protein positive expres-sion and those with negative expression.Conclusion The overexpressions of PIK3CA and p-AKT accompa-nied by the loss of PTEN expression participate in the development and progression of SNSCC,moreover the PIK3CA and p-AKT expressions are related to the poor prognosis of the patients.

Effects of ropivacaine on biological behavior and AMPK/Nrf2 signaling pathway of gastric cancer BGC-823 cells / 国际检验医学杂志

Objective To investigate the effect of ropivacaine on biological behavior of gastric cancer BGC-823 cells and AMP activated protein kinase(AMPK)/nuclear factor E2-related factor 2(Nrf2)signaling path-way.Methods Gastric cancer BGC-823 cells were cultured in vitro.When BGC-823 cells were cultured to log-arithmic growth phase,they were divided into control group(routine culture),cisplatin group(medium con-taining 5 mg/L cisplatin),and low-level(medium containing 25 mg/L ropivacaine),medium-level(medium containing 50 mg/L ropivacaine),and high-level(medium containing 100 mg/L ropivacaine)ropivacaine groups.The cells were cultured for 48 h.Cell counting kit-8(CCK-8)was used to detect the survival rate of BGC-823 cells.Transwell chamber assay was used to detect the invasion ability of BGC-823 cells.The apopto-sis rate of BGC-823 cells was detected by flow cytometry.The mRNA levels of AMPK and Nrf2 in BGC-823 cells were detected by real-time fluorescence quantitative PCR(qPCR).The protein levels of AMPK and Nrf2 in BGC-823 cells were detected by Western blot.Results Compared with the control group,the survival rate,invasion number,AMPK and Nrf2 mRNA and protein levels of BGC-823 cells in the cisplatin group and the low-level,medium-level and high-level ropivacaine groups were decreased(P＜0.05),while the apoptosis rate was increased(P＜0.05).Compared with the cisplatin group,the survival rate,invasion number,AMPK and Nrf2 mRNA and protein levels of BGC-823 cells in the low-level,medium-level and high-level ropivacaine groups were increased(P＜0.05),while the apoptosis rate was decreased(P＜0.05).Compared with the low-level ropivacaine group,the survival rate,invasion number,AMPK,Nrf2 mRNA and protein levels of BGC-823 cells in the medium-level and high-level ropivacaine groups were decreased in oder(P＜0.05),while the apop-tosis rate was increased in oder(P＜0.05).Conclusion Ropivacaine could inhibit the survival and invasion of BGC-823 cells and promote the apoptosis of BGC-823 cells,which may be related to the inhibition of AMPK/Nrf2 signaling pathway.