Effect of Faeces Bombycis Extract on Cajal Interstitial Cells in Diabetic Gastroparesis Rats Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic mechanism of Faeces Bombycis on diabetic gastroparesis （DGP） rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein （PI3K/Akt/mTOR） signaling pathway. MethodDGP rat model was prepared by random selection of 15 out of 105 rats as blank group. The rats successfully constructed were randomly divided into model group， high-，medium- and low- dose groups （3.2， 1.6， 0.8 g·kg-1） and moxapride group （1.5 mg·kg-1）， with 12 rats in each group， and were given gavage for 4 weeks. The gastric emptying rate and random blood glucose were measured. The morphological changes of gastric antrum were observed by hematoxylin-eosin （HE） staining， and the expression of the c-Kit gene was analyzed by immunohistochemistry. The apoptosis of Cajal interstitial cells was observed by in situ end labeling （TUNEL） staining， and the protein expressions of PI3K， phosphorylation（p）-PI3K， Akt， p-Akt， mTOR， and p-mTOR were detected by Western blot. ResultCompared with the blank group， the gastric emptying rate of the model group decreased significantly （P<0.01）， and the glandular structure of the gastric antrum was destroyed. The expression of c-Kit decreased （P<0.01）， and the apoptosis of Cajal interstitial cells （ICC） increased. Compared with the model group， the gastric emptying rate in the high， middle， and low-dose groups of Faeces Bombycis extract and mosapride group increased significantly （P<0.01）. The glandular structure of the gastric antrum became closer， and the apoptosis of ICC decreased. The expression of c-Kit in the high dose group of Faeces Bombycis extract increased significantly. After Western blot testing， compared with the blank group， the protein expression of p-Akt/Akt， p-PI3K/PI3K， and p-mTOR/mTOR in the model group increased. Compared with the model group， the protein expression of p-Akt/Akt in the high dose group of Faeces Bombycis extract decreased （P<0.01）， and the protein expression of p-PI3K/PI3K decreased in the middle and low dose groups of Faeces Bombycis extract and mosapride group decreased （P<0.05， P<0.01）. The protein expression of p-mTOR/mTOR decreased in the low dose group of Faeces Bombycis extract （P<0.05）. In terms of random blood glucose， compared with the blank group， the random blood glucose in the model group increased significantly （P<0.01）， and compared with the model group， the random blood glucose in the high and middle dose groups of Faeces Bombycis extract decreased significantly （P<0.05）. Compared with mosapride group， the protein expression of p-Akt/Akt decreased in the high dose group of Faeces Bombycis extract （P<0.05）， and the protein expression of p-PI3K/PI3K increased in the high， middle， and low dose groups of Faeces Bombycis extract （P<0.05， P<0.01）. ConclusionFaeces Bombycis extract can increase gastric emptying rate， reduce ICC apoptosis， and lower random blood glucose in DGP rats. The high dose group of Faeces Bombycis extract has a significant effect on inhibiting ICC apoptosis， and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

The technologies of small molecule ligand-target protein interaction and high throughput ligand screening / 药学学报

The interaction of drug and target protein is a critical part of new drug discovery. It is the premise for drugs to exert therapeutic effects by targeting specific binding sites of target proteins and thereby affecting its pharmacological activity. Currently, a variety of techniques are exploited to detect the interaction between drug ligands and target proteins. For example, cellular thermal shift assay (CETSA) and differential scanning fluorimetry (DSF) based on thermodynamics, mass spectrometry and nuclear magnetic resonance technology, etc. In addition, high-throughput ligand screening technology provides technical convenience for the search of specific ligand, and is a powerful tool to efficiently identify the interaction between drug ligand and target protein. Here, we summarize the detection techniques of interaction between small molecules and target proteins, and discuss the application of high-throughput ligand screening technology in drug research.

Effect of multi-target protein tyrosine kinase inhibitor Ponatinib on proliferation,adhesion and migration ability of human liver cancer cell line SK-Hep-1 / 中国生物制品学杂志

@#Objective To investigate the effect of a multi-target protein tyrosine kinase inhibitor,Ponatinib,on proliferation,homogeneity adhesion and migration ability of human liver cancer cell line SK-Hep-1.Methods SK-Hep-1 cells were cultured routinely and added with 24 tyrosine kinase inhibitors such as Ponatinib respectively,and the effect of Ponatinib on the survival and proliferation of SK-Hep-1 cells was detected by MTT assay.SK-Hep-1 cells were cultured routinely until the fusion degree reached 90%,then added with 0.1,0.5 and 1.0 μmol/L Ponatinib respectively,and the control group(without Ponatinib) was set up.The effect of Ponatinib on adhesion ability of SK-Hep-1 cells was detected by cell slow aggregation assay and dissociation assay,while the effect on migration ability by scratch test,and the effect on E-cadherin protein expression in SK-Hep-1 cells by Western blot.Results All 24 tyrosine kinase inhibitors inhibited SK-Hep-1 cells,among which Ponatinib showed the strongest inhibitory effect with a IC_(50) of(0.288±0.044) μmol/L.Compared with the control group,the number of cell mass(t=16.143,44.002 and 44.853 respectively,each P <0.001) and N_(TC)/N_(TE) [ratio of single cell number(N) after digestion by trypsin containing EDTA(TE) and CaCl_2(TC)](t=4.276,10.625 and 27.571 respectively,each P <0.05) decreased significantly and E-cadherin protein expression increased significantly(t=-3.757,-4.561and-6.922 respectively,each P <0.05) in 0.1,0.5 and 1.0 μmol/L Ponatinib groups;Scratch migration rate significantly decreased in 0.5 and 1.0 μmol/L Ponatinib groups(t=6.272～16.733 respectively,each P <0.01),while there was no significant difference in 0.1 μmol/L Ponatinib group(t=0.473 and 0.872 respectively,each P> 0.05) after 24 h and 48 h of scratch.Conclusion Ponatinib inhibited proliferation and migration of SK-Hep-1 cells and promoted cell adhesion.

Effect and molecular mechanism of mammalian target of rapamycin complex 2 / Akt signaling pathway on 6-hydroxydopamine-treated SH-SY5Y cell model / 解剖学报

[Abstract] Objective To study whether the regulation of mammalian target of rapamycin complex 2(mTORC2) / Akt signaling pathway has a protective effect on SH-SY5Y cell line damaged by 6-hydroxydopamine (6-OHDA), and to clarify its molecular mechanism. Methods SH-SY5Y cells treated with retinoic acid (RA) were given 6-OHDA, mTORC2 signaling pathway inhibitor PP242 and agonist A-443654 respectively. The changes of cell number in each group were investigated by immunofluorescent staining; The total protein was extracted and the expression level and interaction of key proteins in mTORC2 signaling pathway were determined by Western blotting and co-immunoprecipitation (CoIP); The apoptosis rate of cells in each group was detected by flow cytometry. At the same time, the co-culture Parkinson’ s disease (PD) model was made using SH-SY5Y cell line and Bv-2 cell line; MTT colorimetric method was used to detect the cell viability of each group; ELISA was used to detect the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in cell culture supernatant. Results The number of tyrosine hydroxylase(TH) / proliferating cell nuclear antigen (PCNA) / hochest-, TH / 5-bronmo-2’ -deoxyuridine(BrdU) -labeled positive cells in 6-OHDA-lesioned PD cell model group was significantly lower than that in the normal group; The apoptosis rate was higher; The expression of Rictor, p-Akt and regulated in DNA damage and development 1(REDD1) was increased; There was an interaction between Rictor and p-Akt or REDD1; The cell viability was significantly reduced in the co-culture model; the content of TNF-α and IL-β increased in the cell culture supernatant. With further up-regulation of the abovementioned protein expressions, the cell survival, apoptosis and pro-inflammatory cytokine levels in A-443654 group were significantly ameliorated, while PP242 group showed the opposite changes. Conclusion A-443654 activates mTORC2 signaling pathway by p-Akt, which increases the expression of Rictor and REDD1 protein. These changes contribute to the amelioration in cell survival rate, apoptosis rate, and the proliferation and differentiation and decreasion of apoptosis rate of SH-SY5Y cells. These result improved 6-OHDA-induced cell damage and inhibited the release of pro-inflammatory cytokines.

Advances in the preclinical and clinical research of proteolysis targeting chimera / 生物工程学报

Proteolysis targeting chimera (PROTAC) refers to heterobifunctional small molecules that can simultaneously bind an E3 ubiquitin ligase and a target protein, enabling specific degradation of the target protein with the aid of the ubiquitin proteasome system. At present, most PROTAC drugs are in the clinical trial stage, and the ligands are mainly non-covalent compounds. PROTAC drugs have the advantage of overcoming drug resistance and degrading "undruggable" target proteins, but non-covalent ligands could lead to the hook effect that undermines drug efficacy. With its own advantages, covalent ligands can avoid the occurrence of this phenomenon, which is of great help to the development of PROTAC. This review summarizes the progress in preclinical and clinical research and application of PROTAC molecules targeting three different classes of protein targets, including intranuclear, transmembrane, and cytosolic proteins. We also offer perspective discussions to provide research ideas and references for the future development of PROTAC.

Toxicity mechanism of Rhododendri Mollis Flos: based on serum metabolomics and network toxicology / 中国中药杂志

This study aims to explore the toxicity mechanism of Rhododendri Mollis Flos(RMF) based on serum metabolomics and network toxicology. The toxic effect of RMF on normal rats was evaluated according to the symptoms, serum biochemical indexes, and histopathology. Serum metabolomics was combined with multivariate statistical analysis to search endogenous differential metabolites and related metabolic pathways. The toxic components, targets, and signaling pathways of RMF were screened by network toxicology technique, and the component-target-metabolite-metabolic pathway network was established with the help of serum metabolomics. The result suggested the neurotoxicity, hepatotoxicity, and cardiotoxicity of RMF. A total of 31 differential metabolites and 10 main metabolic pathways were identified by serum metabolomics, and 11 toxic components, 332 related target genes and 141 main signaling pathways were screened out by network toxicology. Further analysis yielded 7 key toxic components: grayanotoxin Ⅲ,grayanotoxinⅠ, rhodojaponin Ⅱ, rhodojaponin Ⅴ, rhodojaponin Ⅵ, rhodojaponin Ⅶ, and kalmanol, which acted on the following 12 key targets: androgen receptor(AR), albumin(ALB), estrogen receptor β(ESR2), sex-hormone binding globulin(SHBG), type 11 hydroxysteroid(17-beta) dehydrogenase(HSD17 B11), estrogen receptor α(ESR1), retinoic X receptor-gamma(RXRG), lactate dehydrogenase type C(LDHC), Aldo-keto reductase(AKR) 1 C family member 3(AKR1 C3), ATP binding cassette subfamily B member 1(ABCB1), UDP-glucuronosyltransferase 2 B7(UGT2 B7), and glutamate-ammonia ligase(GLUL). These targets interfered with the metabolism of gamma-aminobutyric acid, estriol, testosterone, retinoic acid, 2-oxobutyric acid, and affected 4 key metabolic pathways of alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism, steroid hormone biosynthesis, and retinol metabolism. RMF exerts toxic effect on multiple systems through multiple components, targets, and pathways. Through the analysis of key toxic components, target genes, metabolites, and metabolic pathways, this study unveiled the mechanism of potential neurotoxicity, cardiotoxicity, and hepatotoxicity of RMF, which is expected to provide a clue for the basic research on toxic Chinese medicinals.

Research progress on the role of autophagy pathway in acute renal injury and targeted treatment strategies / 中国综合临床

Autophagy is a highly conserved intracellular catabolic process used to degrade cytoplasmic components. In recent years, it has attracted much attention because of its importance in the pathogenesis and targeted therapy of acute and chronic kidney disease. Autophagy plays an important role in maintaining renal homeostasis under physiological and pathological conditions. The study of conditional autophagy related gene knockout specific to various renal cells has gradually revealed the role of autophagy in renal diseases. Recent studies have found that autophagy deficiency may play a key role in different pathological states of the kidney. Activated autophagy shows cytoprotective function in both glomerulus and renal tubulointerstitium, suggesting that the up regulation of autophagy may become a potential therapeutic strategy. However, there is also contrary evidence that autophagy may be harmful, which poses a great challenge to the development of therapeutic strategies for up-regulated autophagy.

Effect of Guizhi Fulingwan on Ovulation Dysfunction in PCOS-IR Rats by Regulating PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of Guizhi Fulingwan on ovulation dysfunction in rats with polycystic ovary syndrome with insulin resistance （PCOS-IR） induced by letrazole combined with high fat emulsion. Method:A total of 72 female SD rats were randomly divided into control group， model group， metformin group and Guizhi Fulingwan low， medium and high dose groups， with 12 rats in each group. Except for control group， rats were given letrozole 0.001g·kg^-1 combined with high-fat emulsion 15 mL·kg^-1 for 21 consecutive days to establish model of PCOS-IR. Guizhi Fulingwan low， medium and high-dose groups were administrated with Guizhi Fulingwan 0.31， 0.62， 1.24 g·kg^-1 respectively， metformin group was administrated with metformin 0.27 g·kg^-1， control group and model group were administrated with 12 mL·kg^-1 of normal saline daily for 30 days. Hematoxylin-eosin（HE） staining was used to observe ovarian tissue pathology morphology， and enzyme-linked immunoassay method （ELISA） was used to detect serum follicle stimulating hormone （FSH）， luteinizing hormone （LH）， testosterone （T）， fasting insulin （FINS） level，and LH/FSH and insulin resistance index （HOMA-IR） were calculated． Western blot was used to detect the expression levels of autophagy key molecular Atg6 yeast homologue （Beclin-1）， autophagy related gene 5（Atg5）， microtubule associated protein light chain 3 （LC3） Ⅱ proteins in the phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein （PI3K/Akt/mTOR） signaling pathway and autophagy related indicators in rat ovarian tissue. Beclin-1 and LC3Ⅱ protein expressions were detected by immunohistochemistry （IHC）. Result:Compared with control group， the thickness of follicles and follicular granulosa cells in the ovary of the model group also decreased， and the number of corpus luteum significantly decreased， while the white membrane thickness of the ovary increased， and the number of atresia follicles and cystic dilatation follicles increased significantly. Serum T， LH， LH/FSH， FINS， FINS， HOMA-IR were significantly increased （<italic>P</italic><0.01）. Phosphorylated （p） -PI3K， p-Akt， and p-mTOR proteins in ovarian tissue were all decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The relative expression levels of autophagy-related protein LC3Ⅱ and Beclin-1 were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with model group， the number of follicles in the low， medium and high dose Guizhi Fulingwan group and the metformin group decreased， the number of follicles in atresia and atresia increased， and the follicular granulosa cell layer thickness increased. Serum T， LH， LH/FSH， FINS and HOMA-IR of Guizhi Fulingwan group were significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， and serum FINS and HOMA-IR of metformin group were significantly decreased （<italic>P</italic><0.01）. The expressions of p-PI3K， p-Akt， and p-mTOR proteins were increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of LC3Ⅱ， Atg5 and Beclin-1 in the medium and high dose groups were significantly decreased （<italic>P</italic><0.01）. Conclusion:Guizhi Fulingwan can activate the PI3K/Akt/mTOR signaling pathway of granular cells， inhibit excessive autophagy of granular cells， improve ovarian function and insulin resistance， and restore ovulation， and the effect is better with high dose.

Effect of Qiyu Sanlong Decoction in Inducing Autophagy of A549 Cells by Regulating Molecular Expression Related to Signal Axis of mTOR-Beclin1-LC3 / 中国实验方剂学杂志

Objective:To observe the effect of Qiyu Sanlong decoction （QYSL） on the expressions of key molecules in signal axis of mammalian rapamycin target protein （mTOR）/yeast Atg6 homologous （Beclin1）/ microtubule-associated protein1 light chain3 （LC3） in A549 cells. Method:With A549 cells as the research object， the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8 （CCK-8） method. The effect of QYSL decoction on A549 cell apoptosis， autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） method， transmission electron microscope （TEM）， Real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group， the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased （P<0.01）， and the formation of autophagosome was significantly increased. Compared with the blank serum group， the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased （P<0.01）， while mRNA and protein expressions of Beclin-1， autophagy related genes 5 （ATG5）， autophagy related genes 13 （ATG13） were significantly increased （P<0.01）. Conclusion:QYSL decoction can induce autophagy in A549 cells， and its specific mechanism may be related to the down-regulation of mTOR expression， the up-regulation of Beclin1， ATG5， ATG13 and LC3 expression， and the promotion of LC3Ⅰ conversion to LC3Ⅱ.

Preventive effect of trace insulin on cognitive dysfunction induced by sevoflurane inhalation anesthesia in newborn rats and its possible mechanism / 解剖学报

Objective To study the preventive effect of microinsulin on cognitive dysfunction induced by sevoflurane inhalation anesthesia in rats and its possible mechanism. Methods Sixty newborn rats were randomly divided into control group (CON), insulin prevention low dose group (LIP), insulin prevention hihg dose group (HIP), and sevoflurane model group (MOD). The prevention group and the model group were induced by sevoflurane to construct rat cognitive dysfunction model. Morris water maze directional sailing test and space exploration test were used to evaluate the learning and memory function of rats; HE staining was used to observe the pathological morphological changes of hippocampus in rats; Flow cytometry was used to detect the hippocampus of rats cell apoptosis; Detection of rapamycin target protein (mTOR) and eukaryotic peptide chain elongation factor 2 (eEF-2) mRNA levels in hippocampus by RT-PCR; The expression levels of brain-derived neurotrophic factor (BDNF), post-synaptic dense protein-95 (PSD-95), synapsin- I, and calmodulin kinase II α (CaMK II α), mTOR and eEF-2 protein were detected by Western blotting. Results The result of the Morris water maze experiment showed that insulin significantly reduced the escape latency and swimming distance of rats, and increased the number of crossing platforms; Flow cytometry result showed that the insulin prevention group significantly inhibited the apoptosis of rat brain neurons, and the inhibition effect of high-dose insulin prevention group was more obvious; RT-PCR and Western blotting analysis found that the expression levels of mTOR and eEF-2 mRNA and proteins in the hippocampus of the model group increased significantly, while the expression levels of BDNF, PSD-95, synapsin- I, and CaMK II α proteins reduced significantly. The expression levels of mTOR and eEF-2 mRNA and proteins in the hippocampus of rats in the insulin prevention group decreased significantly, while the expression levels of BDNF, PSD-95, synapsin- I, and CaMK II α protein increased significantly. The difference was statistically significant (P < 0.05). Conclusion Trace insulin can increase the expression of synapse-related proteins in the hippocampus of cognitive dysfunction rats, reduce their mTOR and eEF-2 mRNA expression levels, and prevent sevoflurane-induced cognitive impairment in rats. The mechanism may be related to the regulation of mTOR-eEF-2 approach.

Based on PI3K/Akt/mTOR Signaling Pathway to Explore Mechanism of Zhigancao Tang Against MIRI-induced Ventricular Tachycardia and Ventricular Fibrillation in Rats / 中国实验方剂学杂志

Objective:Through phosphatidylinositol 3-kinase（PI3K）/protein kinase B （Akt）/mammalian rapamycin target protein （mTOR） signaling pathway， explore the effect of Zhigancao Tang on myocardial ischemia-reperfusion injury（MIRI）The role and mechanism of arrhythmia（ventricular tachycardia and ventricular fibrillation）. Method:The 72 SD rats were randomly divided into sham operation group，model group， Zhigancao Tang low，medium and high dose group（11.43，22.86，45.72 g·kg-1），Wenxin granule group（2.43 g·kg-1），continuous drug intervention for 10 days. Two hours after the last administration，the MIRI model of rat was prepared by ligating the left anterior descending coronary artery，and the changes of electrocardiogram were recorded. After successful modeling，blood and heart tissue were collected to detect the content of creatine creatine（CK），lactate dehydrogenase（LDH）and aspartate aminotransferase（AST）in the serum, the enzyme-linked immunoassay（ELISA） method was used to detect cardiac troponin（CtnI）content, immunohistochemical detection of myocardial PI3K，Akt，mTOR expression. Western blot was used to detect the myocardial autophagy-related protein microtubule-associated protein 1 light chain 3（LC-3），autophagy markers Beclin1 and PI3K/Akt/mTOR signaling pathway related protein expression and phosphorylated p-PI3K，p-Akt，p-mTOR levels. Result:In model group， 100% of ventricular tachycardia and 91.67% of ventricular fibrillation occurred. Compared with sham operation group， the serum levels of CK，LDH，AST，and CtnI in the model group were significantly increased（P<0.01），PI3K，Akt，mTOR AOD values in myocardial tissue were significantly increased （P<0.01），the relative expression of the ratio of LC3-Ⅱ/LC3-Ⅰ and Beclin1 was significantly increased（P<0.01），p-PI3K/PI3K，p-Akt/Akt，and p-mTOR/mTOR were significantly reduced （P<0.01）. Compared with model group， the incidence of ventricular tachycardia and ventricular fibrillation in the high-dose Zhigancao Tang group was significantly reduced （P<0.01），and the duration was the shortest compared with other administration groups（P<0.01）， CK，LDH，AST level and CtnI content were significantly reduced（P<0.01）， the expression of PI3K，Akt and mTOR of Zhigancao Tang group was significantly decreased with increasing dose（P<0.01）， the expression of LC-3 and Beclin1 was accompanied by Zhigancao Tang increase of each dose group of soup had different degrees of decrease （P<0.01），while the expression ratio of PI3K/Akt/mTOR-related protein was significantly increased （P<0.05，P<0.01）. Conclusion:Pretreatment of Zhigancao Tang can reduce the abusually elevated cardiac enzymes CK, LDH, AST and CtnI, inhibit excessive autophagy of cells, and up-regulate the expression of PI3K, Akt and mTOR, indicating that the anti-MIRI arrhythmia effect of Zhigancao Tang may be related to the PI3K/Akt/mTOR signaling pathway.

Effect of Modified Shengjiangsan Influences on PI3K/Akt/mTOR Signaling Pathway and Autophagy in Rats with Membranous Nephropathy / 中国实验方剂学杂志

Objective:To explore the intervention effect of modified Shengjiangsan in rats with membranous nephropathy (MN) and its related mechanism. Method:Rats were injected with cationized bovine serum Albumin (C-BSA) in the tail vein to establish a rat model of membranous nephropathy. The normal group, model group, modified Shengjiangsan group (27.3 g·kg-1) and benazepril group (10 mg·kg-1) were established in this study. Each group was given corresponding dosage of the drug once a day for 4 weeks of continuous intervention. After the administration, the levels of 24-hour urine protein (UTP), total cholesterol (TC), triglyceride (TG), total protein (TP), Albumin (Alb), creatinine (SCr), urea nitrogen (BUN) level was detected. we observed the pathological changes of rat kidneys by the technology of Masson staining, silver hexylamine iodate (PASM) staining and transmission electron microscopy. immunofluorescence technology was used to detect immunoglobulin （Ig）G deposition in rat kidneys. Western blot was used to detect the expression levels of key proteins in phosphatidylinositol 3-kinase/proline protein kinase B/rapamycin target protein (PI3K/Akt/mTOR) signaling pathway and autophagy marker proteins LC3 and Beclin1. Result:Compared with normal group, the UTP, serum TC and TG levels were significantly increased, TP and Alb levels were significantly reduced in model group(P<0.05). We detected the kidney pathological changes include of glomerulus enlargement, basement membrane thickening,vacuolar degeneration, pheotropin deposition, glomerular capillary loop IgG diffuse deposition, electron dense deposits of varying sizes and podocytes under the epithelium extensive integration of foot processes， the expression of p-PI3K, p-Akt and p-mTOR protein was significantly increased (P<0.05). The expression of autophagy marker proteins LC3 and Beclin1 protein decreased significantly(P<0.05). Compared with model group, the UTP, serum TC and TG levels were decreased in the benazepril group and modified Shengjiangsan group, and the TP and Alb levels were increased (P<0.05)， the histopathological changes of rat kidney were all reduced， the expression of p-PI3K, p-Akt and p-mTOR protein was significantly reduced(P<0.05)， autophagy marker proteins LC3 and Beclin1 protein expression were significantly increased. Conclusion:Modified Shengjiangsan can reduce urinary protein, reduce kidney pathological damage and delay disease progression, which is related to its inhibition of PI3K/Akt/mTOR signaling pathway and activation of renal autophagy.

Effects of propofol on TGF-β1 induced activation of hepatic stellate cells and its mechanism / 中国应用生理学杂志

To observe the effects of propofol on the activation of hepatic stellate cell line HSC2-T6 induced by transforming growth factor-beta 1 (TGF-β1) and explore its possible mechanism. The cells were divided into control group, TGF-β1 group, propofol group, TGF-β1 + propofol group, rapamycin group, TGF-β1 + propofol + rapamycin group. Cells were treated with rapamycin (5 μmol/L) for 1 hour, propofol (100 μmol/L) for 1 hour, then TGF-β1 (5 ng/ml) was added to co-culture for 24 hours. Cell proliferation was measured by MTT assay. The concentrations of hyaluronic acid (HA), collagen IV (IV-C) and laminin (LN) in the supernatant of cell culture medium were measured by ELISA. The ultrastructure of cells was observed by transmission electron microscopy. The expressions of alpha-smooth muscle actin (α-SMA), mammalian rapamycin target protein (mTOR), phosphorylated mTOR (p-mTOR) and the autophagy related gene Beclin 1, LC3 and p62 were measured by Western blot. Compared with control group, cell proliferation, the expression of α-SMA, the concentrations of HA, IV-C and LN in culture supernatant, the number of autophages, the expressions of Beclin-1 and LC3-II, the ratio of LC3-II/LC3-I in HSC2-T6 cells were increased significantly, while the expression of p-mTOR, the ratio of p-mTOR/mTOR and the expression of p62 protein were decreased significantly in TGF-β1 group (All P＜0.05). Compared with TGF-β1 group, cell proliferation, the expression of α-SMA, the concentrations of HA, IV-C and LN in culture supernatant, the number of autophages, the expressions of Beclin-1 and LC3-II, the ratio of LC3-II/LC3-I in HSC2-T6 cells in TGF-β1 group were decreased significantly, and the expression of p-mTOR, the ratio of p-mTOR/mTOR and expression of p62 protein were increased significantly in TGF-β1 + propofol group (All P＜0.05). Propofol inhibits the activation of hepatic stellate cells induced by TGF-beta 1, and its mechanism involves the mTOR-autophagy pathway.

Sub-chronic aluminum exposure inhibits synaptic plasticity in hippocampus of rats via inhibiting PI3K/AKT/mTOR signaling pathway / 中国职业医学

OBJECTIVE: To study the effect of sub-chronic aluminum exposure on synaptic plasticity in the hippocampus of rats and to explore the mechanism of phosphatidylinositol 3 kinase(PI3 K)/protein kinase B(AKT)/rapamycin target protein(mTOR) signaling pathway. METHODS: Specific pathogen free adult healthy male SD rats were randomly divided into control group and low-, medium-and high-dose groups based on body weight, with 10 rats in each group. Rats were treated with maltol aluminum solution at the concentrations of 0, 10, 20 and 40 μmol/kg body weight by intraperitoneal injection, 5 days per week for 3 months. After the exposure, rats were weighed. Morris water maze was used to test the learning and memory ability, and the two-electrode binding technique was used to record the long-term potentiation(LTP) amplitude in the hippocampus CA1 area of rats. The protein expression of PI3 K, AKT and mTOR in rat hippocampus tissues was detected by Western blot. RESULTS: After the exposure, the body weights of rats in the medium-and high-dose groups were lower than that of the control group(P<0.05). The results of the positioning navigation experiment showed that the escape latencies of the rats in the medium-and high-dose groups were shorter than that in the control group during the 2 nd to 4 th days of the experiment(P<0.05). The results of space exploration experiments showed that there was no statistical difference on the target quadrant retention time and the number of crossing the platform among the 4 groups(P>0.05). At 1, 30, and 60 min after high-frequency stimulation, the LTP amplitudes in the hippocampus CA1 area of the aluminum-treated groups were lower than that of the control group at the same time point(P<0.05), and the LTP amplitudes of hippocampus CA1 area of rats decreased with the increase of maltol aluminum exposure dose(P<0.01). The relative expression of PI3 K, AKT and mTOR protein in the hippocampus tissues of the aluminum-treated groups was lower than that of the control group(P<0.05), and the relative expression of the above three proteins decreased with the increase of the maltol aluminum exposure dose(P <0.01). CONCLUSION: Sub-chronic aluminum exposure could lead to dose-dependent inhibition of hippocampus synaptic plasticity in rats, thereby impairing the spatial learning ability of rats. This process may be related to inhibition of PI3 K/AKT/mTOR signaling pathway by aluminum.

The role of the mTORC1 signaling pathway during osteogenic differentiation of mouse bone marrow mesenchymal cells under tension stress / 口腔疾病防治

Objective@# To investigate the expression of the mTORC1 signaling pathway during the osteogenic differentiation of mouse bone marrow mesenchymal cells (BMMSCs) under cyclic uniaxial tension and explore its possible role.@*Methods @# The BMMSCs of mice were affected by uniaxial dynamic tensile force. Western blot was used to detect the expression changes of major molecules (mTOR, Raptor, S6K) in the endogenous mTORC1 signaling pathway at 0, 1, 2, 4, and 8 hours after stretching. Chemical colorimetry, ELISA and PCR were used to detect alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2 mRNA, respectively. Then, inhibition, activation and control groups were established by administration of the drugs PP242, MHY1485 and PBS, respectively. Two hours after the stress, the expression of S6K was detected by western blot, and the expression of the osteogenic signal was continuously detected by the above methods.@*Results @#Western blot analysis showed that the main molecules of the mTORC1 signaling pathway were all expressed within 8 hours after traction, and the highest expression was 2 hours after the stress. Compared with those in the control group, the ALP activity and OCN expression decreased and the Runx2 mRNA levels increased after the mTORC1 signal pathway was inhibited (P ＜ 0.001); ALP activity and OCN expression increased after the mTORC1 signal pathway was activated, while the Runx2 mRNA levels decreased (P ＜ 0.001). @*Conclusion @#The mTORC1 signaling pathway participates in the osteogenic differentiation of mouse BMMSCs under tension. The osteogenesis of BMMSCs under cyclic uniaxial tension would be enhanced if the mTORC1 signaling pathway was activated.

Degradation of proteins by PROTACs and other strategies

Blocking the biological functions of scaffold proteins and aggregated proteins is a challenging goal. PROTAC proteolysis-targeting chimaera (PROTAC) technology may be the solution, considering its ability to selectively degrade target proteins. Recent progress in the PROTAC strategy include identification of the structure of the first ternary eutectic complex, extra-terminal domain-4-PROTAC-Von-Hippel-Lindau (BRD4-PROTAC-VHL), and PROTAC ARV-110 has entered clinical trials for the treatment of prostate cancer in 2019. These discoveries strongly proved the value of the PROTAC strategy. In this perspective, we summarized recent meaningful research of PROTAC, including the types of degradation proteins, preliminary biological data in vitro and in vivo, and new E3 ubiquitin ligases. Importantly, the molecular design, optimization strategy and clinical application of candidate molecules are highlighted in detail. Future perspectives for development of advanced PROTAC in medical fields have also been discussed systematically.

Mechanism of Yang-tonifying herbs distributing along kidney meridians in molecular level by network pharmacology / 中草药

Objective: To predict the unique mechanism of Yang-tonifying herbs distributing along kidney meridians in molecular level through network pharmacology technology. Methods: Eight kidney-yang tonifying herbs with common clinical effects and clear therapeutic effects were selected in study. The chemical ingredients of traditional Chinese medicines were searched by TCMSP database. OB and DL values were applied to screen the active substance and the chemical similarity target prediction methods of Pub Chem database were used to predict the target proteins of TCM; The PPI between the target proteins of the kidney-yang tonifying herbs and the KEGG signal pathway were searched by the STRING database; The nodes in the PPI network were evaluated by the weighted PageRank algorithm and then the core target protein was screened. Using the Cytoscape 3.6.0 software, a compound-target network, a herb-target-PPI network, and a target-pathway network were constructed. Results: Through the network analysis, 21 key targets and 40 signal pathways of the kidney-yang tonifying herbs were screened. The medicinal played the role of warming and tonifying kidney-yang by T cell regulation, sex hormone regulation, immune response, and delaying aging. The mechanism may be related to thyroid hormone signaling pathway, neurotrophin signaling pathway, TNF signaling pathway and estrogen signaling pathway. Conclusion: The method based on network pharmacology could help to find the key targets and signal pathways of the kidney-yang tonifying herbs, which provides useful information and data support for further interpretation of the classification meaning of the kidney-yang tonifying herbs in TCM

Study on Potential Mechanism of the Seed of Draba nemorosa Based on Network Pharmacology / 中国药房

OBJECTIVE： To investigate the potential pharmacological mechanism of the seed of Draba nemorosa， and to provide reference for further development， utilization and clinical application. METHODS： Effective components and related target proteins of D. nemorosa were screened and identified by using TCMSP and STRING database. Cytoscape 3.7.0 software was used to construct a visual network of effective components and target proteins for the seed of D. nemorosa， and the network topology analysis was performed. The targeting protein-protein interaction （PPI） network was constructed and analyzed by STRING database and Cytoscape 3.7.0 software. KEGG pathway enrichment of target proteins was analyzed by DAVID bioinformatics resource database. RESULTS： A total of 9 effective components were screened from the seed of D. nemorosa， including quercetin， kaempferol， β-sitosterol， etc. Totally 174 target proteins were obtained， mainly including PTGS2， NCOA2， PGR， etc. Among them， JUN and MAPK1 were core proteins in PPI network. KEGG enrichment pathway included PI3K/Akt signaling pathway， TNF-α signaling pathway， HIF-1 signaling pathway， Toll-like receptor signaling pathway and thyroid hormone signal pathway， etc. CONCLUSIONS： Effective components from the seed of D. nemorosa such as quercetin， kaempferol and β-sitosterol may act on PTGS2， JUN and MAPK1 target proteins through PI3K-Akt signaling pathway and TNF-α signaling pathway， thus exert the effects of purging lung， relieving asthma， promoting edema and reducing edema.

Study on Virtual Screening of Active Components from Yiqi Ziyin Decoction in the Treatment of Type 2 Diabetes Mellitus / 中国药房

OBJECTIVE： To conduct virtual screening for active component， target and pathway of TCM Yiqi ziyin decoction in the treatment of type 2 diabetes mellitus， and to elucidate the pharmacodynamic mechanism of this decoction in the treatment of type 2 diabetes mellitus at molecular level so as to provide theoretical basis for its popularization， application and rational organization. METHODS： Chemical compounds isolated from 10 ingredients of Yiqi ziyin decoction were retrieved from Handbook of Chemical Constituents of Original TCM Plants. Drug/drug-like molecules for the treatment of type 2 diabetes mellitus were retrieved from DrugBank database. Three-dimensional molecular structures of each component were downloaded from PubChem Compound database. The structure was optimized by using Cerius2 4.10 software， and the small molecular data set of Yiqi ziyin decoction was established. Using “type 2 diabetes mellitus” as keywords， target protein of type 2 diabetes mellitus were retrieved and screened from target protein databases， and their three-dimensional structures were downloaded from protein database. The selected compound molecules （ligands） were docked with target proteins （receptors）， and small molecules with high activity （sorted according to the values of Degree and Betweenness） and target proteins with high correlation were found. The interaction mechanism between ligand and receptor was discussed by taking the fine docking of selected small molecules with target protein （N-trans-caffeoyl tyrosine molecule and glycogen synthase kinase-3β） as an example. RESULTS： There were 732 kinds of chemical components and 127 drug/drug-like molecules； 30 target proteins of type 2 diabetes mellitus were screened. According to the values of Degree and Betweenness， 8 molecules with high activity （such as N-trans-caffeoyl tyrosine， hyperin and so on） and 10 target proteins with high correlation （such as carbonic anhydrase1， glycogen synthetase kinase-3β and so on） were screened out. The interaction between N-trans-caffeoyl tyrosine and glycogen synthase kinase-3β could form hydrogen bonds and π alkylation， thus affecting the structural stability and activity of the latter and reducing blood glucose. CONCLUSIONS： In this study， it found there existed not only one molecule compound interacting with multiple targets， but also different molecule compound  interacting with the same target on Yiqi Ziyin decoction for the treatment of type 2 diabetes mellitus by virtual screening， it revealed the characteristics of multi-component and multi-target synergism of TCM prescriptions at the molecular level， providing reference for screening lead compounds and structural modification of new drug for diabetes mellitus.

Study on Anti-inflammatory Mechanism of Couplet Medicine of Notopterygium incisum -Angelica pubescens Based on Network Pharmacology / 中国药房

OBJECTIVE： To predict the anti-inflammatory active components and mechanism of couplet medicine of Notopterygium incisum-Angelica pubescens. METHODS： According to the principle of oral bioavailability≥30％ and drug- likeness≥0.18， active components of N. incisum and A. pubescens were screened； TCMSP was used to predict and screen the potential target of them. Using “Anti-inflammatory” as keyword， inflammatory related target genes were retrieved from human gene database Genecards. Common target was screened by mapping the target genes of active ingredients from couplet medicine of N. incisum-A. pubescens. The active ingredient-target network was established by using Cytoscape 3.5.1 software. The screened targets were used to construct the target protein interaction （PPI） network on the STRING V 10.5 platform. Its anti-inflammatory mechanism was studied by KEGG signaling pathway and GO biological enrichment analysis. RESULTS： Totally 15 active components such as coumarin， beta-sitosterol， ammidin， nodakenin were selected from couplet medicine of N. incisum-A. pubescens. Acting on 49 targets such as transcription factor AP-1， PI3-kinase subunit gamma， estrogen receptor， they mainly involved 19 signaling pathways such as hepatitis B and cell apoptosis， and were involved in 47 biological processes such as regulating inflammatory response and prostaglandin biosynthesis. CONCLUSIONS： The anti-inflammatory mechanism of active components of couplet medicine of N. incisum-A. pubescens on multi-target， multi-channel and multi-biological processes is predicted， and it points out the direction for further anti-inflammatory mechanism study.