Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

Small Molecules Accelerate Skin Wound Healing: Shikonin Efficacy and Mechanism of Action in Mice / Las Moléculas Pequeñas Aceleran la Curación de Heridas en la Piel: Eficacia de Shikonin y Mecanismo de Acción en Ratones

SUMMARY: The objective of this study was to investigate the therapeutic wound healing potential and molecular mechanisms of shikonin as small molecules in vitro. A mouse burn model was used to explore the potential therapeutic effect of shikonin; we traced proliferating cells in vivo to locate the active area of skin cell proliferation. Through the results of conventional pathological staining, we found that shikonin has a good effect on the treatment of burned skin and promoted the normal distribution of skin keratin at the damaged site. At the same time, shikonin also promoted the proliferation of skin cells at the damaged site; importantly, we found a significant increase in the number of fibroblasts at the damaged site treated with shikonin. Most importantly, shikonin promotes fibroblasts to repair skin wounds by regulating the PI3K/AKT signaling pathway. This study shows that shikonin can effectively promote the proliferation of skin cell, and local injection of fibroblasts in burned skin can play a certain therapeutic role.

El objetivo de este trabajo fue investigar el potencial terapéutico de cicatrización de heridas y los mecanismos moleculares de la shikonina como moléculas pequeñas in vitro. Se utilizó un modelo de quemaduras en ratones para explorar el posible efecto terapéutico de la shikonina; Rastreamos las células en proliferación in vivo para localizar el área activa de proliferación de células de la piel. A través de los resultados de la tinción para patología convencional, encontramos que la shikonina tiene un buen efecto en el tratamiento de la piel quemada y promueve la distribución normal de la queratina de la piel en el sitio dañado. Al mismo tiempo, la shikonina también promovió la proliferación de células de la piel en el sitio dañado. Es importante destacar que encontramos un aumento significativo en la cantidad de fibroblastos en el sitio dañado tratado con shikonina. Lo más importante es que la shikonina promueve la función reparadora de fibroblastos en las heridas de la piel regulando la vía de señalización PI3K/ AKT. Este estudio muestra que la shikonina puede promover eficazmente la proliferación de células de la piel y que la inyección local de fibroblastos en la piel quemada puede desempeñar un cierto papel terapéutico.

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.

Study on the effect and mechanism of the alcoholic extract from Scabiosa comosa against hepatic fibrosis / 中国药房

OBJECTIVE To explore the effect and mechanism of the alcoholic extract from Scabiosa comosa against hepatic fibrosis （HF）. METHODS Intragastrical administration of carbon tetrachloride was given to induce HF model. By observing the pathological changes in liver tissue， mRNA and protein expressions of HF indexes [α-smooth muscle actin （α-SMA）， collagen type Ⅰ] and phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） pathway-related factors were detected， and the improvement effects and possible mechanism of low-dose， medium-dose and high-dose （50， 100， 200 mg/kg） of alcoholic extract from S. comosa on HF model rats were investigated. Drug-containing serum was prepared by intragastrical administration of alcoholic extract from S. comosa at a concentration of 1 800 mg/（kg·d） （calculated by the amount of raw material）. The effects of drug- containing serum of alcoholic extract from S. comosa on the expression of miRNA-21 were observed through the intervention of HSC-T6 cells with low， medium and high concentrations of drug-containing serum of alcoholic extract from S. comosa （diluted to 10%， 15%， 20%）. miRNA-21 mimics or inhibitors were used to transfect HSC-T6 cells， and the mRNA and protein expressions of factors related to the PI3K/Akt signaling pathway were detected. RESULTS The results of in vivo experiments showed that low， medium and high doses of alcoholic extract from S. comosa significantly ameliorated the histopathological changes in liver tissue of HF rats， and the percentage of collagen was significantly reduced （P＜0.01）； mRNA and protein expressions of the indicators related to HF as well as PI3K and Akt were significantly reduced （P＜0.01）， and mRNA and protein expressions of phosphatase and tensin homolog deleted on chromosome ten （PTEN） were increased in liver tissue of rats （P＜0.01）. The results of in vitro experiments showed that drug-containing serum of alcoholic extract from S. comosa significantly inhibited the expression of miRNA-21 at low， medium and high concentrations （P＜0.01）； whereas after transfection with miRNA-21 mimics， it was found that miRNA-21 mimics significantly increased mRNA and protein expressions of PI3K and Akt （P＜0.01）， while significantly decreased mRNA and protein expressions of PTEN （P＜0.01）； after transfection with miRNA-21 inhibitor， the changes of above indexes were opposite to the above results （P＜0.01）. CONCLUSIONS Alcoholic extracts of S. comosa may inhibit the PI3K/Akt signaling pathway by affecting the expression of miRNA-21， so as to achieve the effect of anti-hepatic fibrosis.

Effects and mechanism of Setaria italica extract on improving sleep in insomnia mice / 中国药房

OBJECTIVE To investigate the effects of Setaria italica extract on improving insomnia model mice and to explore its potential mechanisms. METHODS The mice were randomly assigned into blank group， model group， positive control group （diazepam， 2.6 mg/kg）， and S. italica extract low-dose， medium-dose and high-dose groups （1.2， 2.4， 4.8 g/kg）， with 10 mice in each group. Except for the blank group， all other groups received intraperitoneal injection of para-chlorophenylalanine （PCPA） to establish the insomnia model. After modeling， the blank group and model group were given a constant volume of normal saline intragastrically， and administration groups were given relevant medicine intragastrically， with a volume of 0.01 mL/g， once a day， for 7 consecutive days. After the administration， the open-field test was conducted to observe the praxiological changes of mice， and to determine the levels of 5-hydroxytryptamine （5-HT） and 5-hydroxyindoleacetic acid （5-HTAA） in the hippocampal tissue， as well as the contents of 5-HT， brain-derived neurotrophic factor （BDNF）， interleukin-2 （IL-2）， IL-6， B-cell lymphoma-2 （Bcl- 2）， and Bcl-2-associated X protein （Bax） in the serum. The expression of phosphoinositide 3-kinase/protein kinase B/nuclear factor- κB （PI3K/Akt/NF-κB） signaling pathway related protein was determined in the hippocampus of mice. RESULTS Compared with the model group， the total exercise time of mice in S. italica extract high-dose group was significantly prolonged， but the total rest time was significantly shortened （P＜0.01）； the number of standing times and modification times were significantly reduced （P＜ 0.01）. The contents of 5-HT， BDNF， and Bcl-2 in serum， and Bcl-2/Bax were significantly increased， while the contents of IL-2， IL-6， and Bax were significantly reduced （P＜0.05 or P＜ 0.01）. The content of 5-HTAA in the hippocampal tissue and 202104010910029）；the phosphorylation levels of PI3K and Akt proteins were increased significantly， while the phosphorylation level of NF-κB p65 protein was decreased significantly （P＜0.05）.CONCLUSIONS High-dose of S. italica extract demonstrates significant therapeutic effects on insomnia in mice， and the mechanism of which may be associated with the regulation of PI3K/Akt/NF-κB signaling pathway.

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.

Jianwei Xiaozhang Tablets Improves Precancerous Lesions of Gastric Cancer in Rats via Regulating PI3K-Akt-eNOS Pathway / 广州中医药大学学报

Objective To investigate the therapeutic effect and mechanism of Jianwei Xiaozhang Tablets on rats with precancerous lesions of gastric cancer(PLGC).Methods Forty male SD rats were randomly divided into the normal group,the model group,the folic acid group and the Jianwei Xiaozhang Tablets group,with 10 rats in each group.In addition to the normal group,the other three groups of rats were prepared by gavage with Ranitidine Aqueous Solution combined with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)solution drinking method for the preparation of PLGC model.After successful modeling,drugs were administered accordingly for 7 weeks.The changes in body mass of rats during modeling and drug administration were recorded,the gross view of the stomach was observed and scored pathologically,the coefficients of spleen and liver were determined,the pathological changes in gastric tissue were observed by hematoxylin-eosin(HE)staining,enzyme-linked immunosorbent assay(ELISA)was used to measure serum gastrin(GAS),motilin(MTL)and glucagon(GC),Alisin Blue-Periodic Acid Schiff's(AB-PAS)staining was used to observe the thickness of the mucosal layer of gastric tissues,the expressions of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),and endothelial-type nitric oxide synthase(eNOS)proteins in gastric tissues were detected by protein immunoblotting(Western Blot),and the expression of vascular endothelial growth factor A(VEGFA)protein in gastric tissues was detected by immunofluorescence staining.Results Compared with the normal group,the body mass of rats in the model group grew slowly during the experimental period,gastric macroscopic pathological scores were significantly increased(P＜0.01),splenic coefficient and hepatic coefficient were significantly decreased(P＜0.01),the gastric tissues showed cuprocyte hyperplasia and intestinal chemotaxis,gastric tissues'inflammation scores were significantly increased(P＜0.01),the serum GAS content was significantly increased(P＜0.01),and the MTL,GC contents were significantly reduced(P＜0.05),and the thickness of the mucous membrane layer of gastric tissue was significantly reduced(P＜0.05),the protein expression levels of PI3K,p-PI3K,Akt,p-Akt and eNOS were reduced(P＜0.01),and the protein expression level of VEGFA was reduced(P＜0.01);compared with the model group,the above indexes of the Jianwei Xiaozhang Tablets group and the folic acid group were all significantly improved(P＜0.05 or P＜0.01),among which,the Jianwei Xiaozhang Tablets group had a better improvement effect in the proliferation of cup cells and intestinal chemotaxis in gastric tissues,the content of serum GAS,and the thickness of the mucous layer in gastric tissues.Conclusion The mechanism of the improvement of PLGC in rats by Jianwei Xiaozhang Tablets may be related to the activation of the PI3K-Akt-eNOS pathway,which in turn promotes the angiogenesis and repair of gastric damaged tissues.

Therapeutic effect of pachymic acid on Helicobacter pylori-associated gastritis in rats by regulating the PI3K/AKT/NF-κB signaling pathway / 基础医学与临床

Objective To explore the therapeutic effect and mechanism of pachymic acid(PA)on Helicobacter py-lori(Hp)-associated gastritis in rats.Methods A rat model of Hp-associated gastritis was established;all rats were separated into control group(CT group),model group(group M),PA low-dose group(PA L group),PA high-dose group(PA H group),and PA H+phosphatidylinositol 3-kinase(PI3K)activator(740 Y-P)group;the gastric mucosal injury index(UI)of rats in each group was evaluated,transmission electron microscopy was applied to observe the morphology of gastric mucosal cells.HE staining was applied to evaluate the pathological characteristics of gastric mucosa.ELISA was applied to detect the levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),IL-10,induc-ible nitric oxide synthase(iNOS),and superoxide dismutase(SOD)in gastric tissue.Western blot method was applied to detect the expression of PI3K,phosphorylated PI3K(p-PI3K),protein kinase B(AKT),p-AKT,nuclear factor(NF)-κB p65,and p-NF-κB p65 proteins.Results Compared with the CT group,the gastric mucosa erosion,epithelial ede-ma,congestion,and severe ulcers were observed in the group M,with epithelial cell pyknosis and inflammatory cell in-filtration,the UI,IL-6,TNF-α,iNOS,and the expression levels of p-PI3K/PI3K,p-AKT/AKT,p-NF-κB p65/NF-κB p65 proteins increased,the levels of IL-10 and SOD decreased(P<0.05);compared with group M,the gastric mucosal damage and inflammatory cell infiltration in the PA L and PA H groups were improved,the UI,IL-6,TNF-α,iNOS by the host animal and the expression of p-PI3K/PI3K,p-AKT/AKT,p-NF-κB p65/NF-κB p65 proteins all decreased,the level of IL-10 and SOD was increased(P<0.05);compared with the PA H group,the pathological damage of the gastric mucosa in the PA H+740 Y-P group was aggravated,with epithelial cell pyknosis.The UI,IL-6,TNF-α,iNOS,and the expression of p-PI3K/PI3K,p-AKT/AKT,p-NF-κB p65/NF-κB p65 proteins increased,the levels of IL-10 and SOD decreased(P<0.05).Conclusions PA might facilitate the treatment of Hp-associated gastritis in rats by inhibiting the PI3K/AKT/NF-κB signaling pathway.

Cadmium promotes senescence of annulus fibrosus cells via activation of PI3K/Akt signaling pathway / 中国组织工程研究

BACKGROUND:Cadmium is a common environmental pollutant,which can damage multiple organs and tissues,such as the kidney and bone,but its effect on annulus fibrosus cells in the intervertebral disc has been less reported. OBJECTIVE:To investigate the effect of cadmium chloride on the senescence of annulus fibrosus cells and the role of PI3K/Akt signaling pathway. METHODS:Annulus fibrosus cells from Sprague-Dawley rat intervertebral discs were harvested and passage 3 cells were intervened with different concentrations of cadmium chloride(0,1,5,10,20 μmol/L).Cell viability and proliferation were detected by cell counting kit-8 assay.Transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis were performed on annulus fibrosus cells with or without cadmium chloride addition.Passage 3 annulus fibrosus cells were divided into control group,cadmium chloride group and LY294002 group.Cell proliferation rate was detected by EdU method,positive cell rate was detected by senescence-associated β-galactosidase staining,and expressions of senescence-associated proteins(p16,p21 and p53)and p-Akt at protein and mRNA levels were measured by western blot,RT-PCR and immunofluorescence. RESULTS AND CONCLUSION:5 μmol/L cadmium chloride could inhibit the proliferation of annulus fibrosus cells.Results from the Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis showed that the main signal transduction pathways included PI3K/Akt,cell cycle and p53 signaling pathways,which were related to cell senescence and proliferation.PI3K/Akt signaling pathways with significant differential expression were selected for validation.Compared with the control group,the EdU-positive rate was significantly decreased in the cadmium chloride group(P<0.05),while the β-galactosidase-positive rate,the expression of senescence-associated proteins(p16,p21 and p53)and p-Akt significantly increased(P<0.05).Compared with the cadmium chloride group,the EdU-positive rate and p-Akt expression were significantly decreased in the LY294002 group(P<0.05),while the β-galactosidase-positive rate and the expression of senescence-associated proteins(p16,p21 and p53)significantly increased(P<0.05).To conclude,cadmium chloride can regulate the senescence of annulus fibrosus cells by activating the PI3K/Akt signaling pathway,thereby inducing the occurrence and progression of intervertebral disc degeneration.

Mechanism by which static exercise improves insulin resistance in skeletal muscle of type 2 diabetes / 中国组织工程研究

BACKGROUND:Skeletal muscle insulin resistance is the key pathological link of type 2 diabetes.Static exercise can effectively improve skeletal muscle insulin resistance,but the mechanism remains unclear. OBJECTIVE:To explore the mechanism of static exercise on insulin resistance in the skeletal muscle of type 2 diabetic mice based on the phosphatidyl inositol 3-kinase(PI3K)/protein kinase B(AKT)/glucose transporter(GLUT4)signaling pathway. METHODS:After 1 week of adaptive feeding,7 out of 40 C57BL/6 mice were randomly selected as blank group and fed common diet,while the other mice were fed high-fat diet and taken to prepare type 2 diabetes models through the low-dose streptozotocin intraperitoneal injection.Twenty-four mice were successfully modeled and they were randomly divided into model group(n=8),metformin group(n=8)and static exercise group(n=8),which continued to be fed high-fat diet.The metformin group was given 200 mg/kg metformin dissolved in normal saline(2 ml/kg)by gavage,once a day,for 6 weeks.The static exercise group was given normal saline daily by gavage and carried out static exercise,30 minutes a day,6 days per week.The model group was given the same dose of normal saline daily by gavage without exercise intervention.After the intervention,the fasting blood glucose of each group was detected,the intraperitoneal glucose tolerance test was performed,and the area under the glycemic curve was calculated.Glycosylated hemoglobin,serum insulin,insulin resistance index were detected by ELISA.Total cholesterol,triglyceride,high-density lipoprotein,low-density lipoprotein were detected using biochemical methods.The mRNA expression levels of PI3K,AKT and GLUT4 in the gastrocnemius of mice were detected by real-time quantitative PCR.Morphological changes of the gastrocnemius were observed by hematoxylin-eosin staining,and the cross-sectional area of muscle fibers was calculated. RESULTS AND CONCLUSION:Compared with the blank group,fasting blood glucose,glycosylated hemoglobin,area under the glycemic curve,insulin resistance index,total cholesterol,triglyceride and low-density lipoprotein levels were significantly increased in the model group(P<0.01,P<0.05).Whereas,these indicators were significantly lower in the static exercise and metformin group than the model group(P<0.01,P<0.05).Compared with the blank group,serum insulin and high-density lipoprotein levels were significantly declined in the model group(P<0.01)and the mRNA expression of PI3K,AKT and GLUT4 in the gastrocnemius of mice were also significantly reduced(P<0.01).These indicators were significantly elevated in the metformin group and static exercise group compared with the blank group(P<0.01).Compared with the blank group,the muscle fibers in the model group were disordered,and the muscle cells atrophied and the muscle fiber gap widened.The cross-sectional area of muscle fibers was significantly decreased in the model group compared with the blank group(P<0.01).Compared with the model group,atrophy of the gastrocnemius fibers and muscle fiber space were improved in the static exercise group and the metformin group,and the cross-sectional area of muscle fiber was significantly increased in both groups(P<0.01).These findings indicate that static resistance training may promote glucose uptake and utilization by up-regulating the expression of PI3K,AKT and GLUT4 mRNA in skeletal muscle tissue,thereby improving the morphology and function of skeletal muscle tissue,alleviating insulin resistance and regulating glucose homeostasis.

Melatonin inhibits hydrogen peroxide-induced injury of human nucleus pulposus cells / 中国组织工程研究

BACKGROUND:Intervertebral disc degeneration is one of the most common underlying factors causing low back pain.Recent studies have shown that melatonin has a positive effect on alleviating intervertebral disc degeneration.However,the underlying mechanism of melatonin remains to be elucidated. OBJECTIVE:To explore the biological effect and potential mechanism of melatonin in inhibiting hydrogen peroxide(H2O2)-induced injury of human nucleus pulposus cells. METHODS:Human nucleus pulposus cells insolated from degenerative intervertebral disc were cultured in vitro.Cell proliferation and the optimal intervention concentration of melatonin and H2O2 were detected by cell counting kit-8.The Human nucleus pulposus cells treated with H2O2 were used as a model group;the cells treated with H2O2 and intervened with melatonin were used as a melatonin group;the cells cultured in simple medium were used as a control group.The reactive oxygen species levels were detected by 2',7'-dichlorofluorescin diacetate(DCFH-DA),the expression levels of BAX and Caspase3 were detected by immunofluorescence,and the mRNA expression levels of BAX,BCL-2,Casepase3,PI3K and AKT were detected using the real-time fluorescent quantitative reverse transcription PCR. RESULTS AND CONCLUSION:The results of cell counting kit-8 experiment showed that the optimal intervention concentration of H2O2 was 400 μmol/L and the optimal intervention concentration of melatonin was 5 μmol/L.The reactive oxygen species level in the melatonin group was significantly lower than that in the model group.The average fluorescence intensity of BAX and Caspase3 in the melatonin group was significantly lower than that in the model group.The mRNA expressions of BAX and Caspase3 in the melatonin group were lower than those in the model group,while the mRNA expression of Bcl-2 was increased.In addition,the mRNA expressions of PI3K and AKT were also higher in the melatonin group compared with the model group.To conclude,melatonin may protect human nucleus pulposus cells from H2O2-induced oxidative damage through the PI3K/AKT signaling pathway.

Extracts of Sambucus adnata Wall.inhibit abnormal angiogenesis in a rat model of osteoarthritis / 中国组织工程研究

BACKGROUND:Previous studies showed that extracts of Sambucus adnata Wall.have the ability to promote the proliferation and differentiation of osteoblasts,fracture healing,anti-inflammatory and antioxidant effects,which can effectively alleviate the development of osteoarthritis.Vascular endothelial growth factor,on the other hand,is a biomarker for the evaluation of osteoarthritis severity. OBJECTIVE:To investigate the effect and mechanism of two extracts of Sambucus adnata Wall.(methanol extract SAW-ME and dichloromethane extract SAW-DCE)on angiogenesis in osteoarthritis. METHODS:(1)Rat models of osteoarthritis were established using anterior cruciate ligament transection and given SAW-ME and SAW-DCE.A sham group was set as a control.Immunohistochemistry and immunofluorescence were used to detect the changes of articular vascular endothelial growth factor A in joint tissue and vascular endothelial growth factor and"H"type blood vessels in serum of osteoarthritis rats.(2)Vascular endothelial cells EA.hy926 were used as the research object and intervened with SAW-ME and SAW-DCE.Cell proliferation was then detected by MTT assay.Vascular endothelial growth factor was used to induce EA.hy926 cells,and the model of angiogenesis was replicated.Cell scratch assay and tube formation assay were performed to study the role and mechanism.(3)EA.hy926 cells were used for transcriptome sequencing to analyze the characteristic changes of cell differential genes and related functions after SAW-DCE intervention. RESULTS AND CONCLUSION:(1)SAW-ME and SAW-DCE downregulated the expression of vascular endothelial growth factor A in the rat knee cartilage and reduced the formation of"H"type vessels in osteoarthritis rats.SAW-ME could significantly decrease the level of vascular endothelial growth factor in serum of osteoarthritis rats(P<0.05).SAW-DCE could also decrease the level of vascular endothelial growth factor in serum of osteoarthritis rats,but there was no significant change.(2)Both SAW-ME and SAW-DCE significantly inhibited vascular endothelial cell migration and tube formation,and downregulated the expression of Ang1 and Tie2 proteins.(3)Transcriptome sequencing analysis found that abnormal angiogenesis in osteoarthritis was related to the PI3K/AKT signaling pathway.(4)To conclude,SAW-ME and SAW-DCE can inhibit angiogenesis in the rat model of osteoarthritis,and the mechanism may be related to the Ang1/Tie2 and PI3K/AKT signaling pathways.

Effect of Wedelolactone on Oxidative Injury in HUVECs Via the PI3K/Akt/mTOR Signaling Pathway / 医药导报

Objective To study the protective effect of Wedelolactone(WEL)against inflammatory injury in human umbilical vein endothelial cells(HUVECs)and its molecular mechanism by inducing PI3K/Akt/mTOR.Methods The model of atherosclerosis(AS)oxidative stress injury in HUVECs was induced with 200 μmol·L-1 of hydrogen peroxide for 24 h.The experimental groups were as follows:normal control group,DMSO(dimethyl sulfoxide)group,H2O2 group,and WEL group.MTT was used to measure the cell survival rate of each group;flow cytometry was used to assess intracellular ROS levels;fluorescence microscopy was used to detect the expression of p62 protein;immunoblotting assay was used to determine the protein expression levels for apoptosis-related proteins associated with PI3K/Akt/mTOR signaling pathway and autophagy-related proteins.Results Compared with the H2 O2 group,the HUVEC cell survival rate was significantly inhibited in the WEL group(P＜0.05).ROS production was significantly lower,and the protein expressions of SOD1 and p62 were significantly increased in the WEL group as compared to the hydrogen peroxide group.The protein expression of p-mTOR,p-Akt,and p-PI3K was significantly decreased in hydrogen peroxide(P＜0.01);In the WEL experiment,p-mTOR,p-Akt,and p-PI3K were increased significantly in the post-injury HUVECs(P＜0.01).Conclusion Wedelolactone inhibits HUVECs'autophagy by suppressing H2O2-induced inflammatory damage in HUVECs,which may be related to the fact that WEL promotes the phosphorylation of PI3K,Akt,and mTOR proteins,inhibits autophagy and thus resists oxidative stress damage in HUVECs cells.

Total flavonoids of Pterocarya hupehensis Skan inhibit migration and in-vasion of fibroblast-like synoviocytes by regulating PI3K/AKT signaling pathway / 中国病理生理杂志

AIM:To observe how total flavonoids of Pterocarya hupehensis Skan(PHSTF)affects the migra-tion and invasion of human rheumatoid fibroblast-like synoviocyte line MH7A.METHODS:The MH7A cells were divided into control group(without any treatment),low-,medium-and high-dose(6.25,12.5 and 25 mg/L,respectively)PHSTF groups,phosphatidylinositol 3-kinase(PI3K)inhibitor 740Y-P(10 μmol/L)group,and 740Y-P(10 μmol/L)+high-dose(25 mg/L)PHSTF group.The viability of the MH7A cells was determined by CCK-8 assay,while the migration and inva-sion were assessed by scratch and Transwell assays.The protein levels of matrix metalloproteinase-2(MMP-2),MMP-9,PI3K,p-PI3K,AKT and p-AKT were detected by Western blot.RESULTS:Compared with control group,the cell via-bility in PHSTF treatment groups was reduced(P<0.05),the cell wound healing area was significantly decreased(P<0.01),migratory and invasive cells in the lower chamber were significantly reduced(P<0.01),and the protein expres-sion of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT were decreased(P<0.01).Compared with high-dose PHSTF group,the addition of PI3K/AKT pathway agonist 740Y-P significantly increased the migration and invasion ability of MH7A cells(P<0.01)and elevated the protein expression of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT(P<0.01)under the treatment with PHSTF.CONCLUSION:Total flavonoids of Pterocarya hupehensis Skan could inhibit the migration and invasion of MH7A cells by regulating the PI3K/AKT signaling pathway.

Role and mechanism of DCLK1 in gastric cancer stem cells / 中国病理生理杂志

AIM:To investigate the effect of doublecortin-like kinase 1(DCLK1)on the biological properties of gastric cancer stem cells,and to explore its possible mechanism.METHODS:Serum-free suspension culture of gastric cancer stem cells and targeted inhibition of DCLK1 activity in gastric cancer stem cells with DCLK1 inhibitor DCLK1-IN-1 were performed.The expression levels of DCLK1,stemness-related proteins(SOX2 and OCT4),proliferation-related pro-teins(cyclin D1 and c-MYC),drug resistance-related proteins(ABCG2 and TOP2A),epithelial-mesenchymal transition-related proteins(E-cadherin,vimentin and Snail),and PI3K/AKT/mTOR signaling pathway-related proteins in gastric cancer stem cells were examined by Western blot.The effects of DCLK1 on viability and drug resistance of gastric cancer stem cells were determined by CCK-8 assay,and the effects of DCLK1 on self-renewal of gastric cancer stem cells were de-termined by methylcellulose spheroid-forming assay.Wound-healing and Transwell assays were performed to assess the ef-fect of DCLK1 on the migration and invasion of gastric cancer stem cells.RESULTS:The expression levels of DCLK1 and stemness-related proteins SOX2 and OCT4 in gastric cancer stem cells were significantly higher than those in parental cells(P＜0.01).The proliferation,drug resistance,migration and invasion of gastric cancer stem cells in DCLK1 inhibi-tion group were significantly lower than those in Sphere cell group(P＜0.01).The expression levels of proliferation-related proteins(c-MYC and cyclin D1)and drug resistance-related proteins(TOP2A and ABCG2)were down-regulated,the ex-pression of epithelial marker E-cadherin was up-regulated,the expression of mesenchymal markers vimentin and Snail was down-regulated,and the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins and their phosphoryla-tion levels were reduced in DCLK1 inhibition group(P＜0.05).CONCLUSION:DCLK1 is highly expressed in gastric cancer stem cells,which may be involved in the proliferation,drug resistance and invasion of gastric cancer stem cells by regulating PI3K/AKT/mTOR signaling pathway.It suggests that DCLK1 can be used as a potential target for gastric cancer stem cells.

Effects of diosgenin on autophagy of human osteosarcoma cells / 中成药

AIM To investigate the effects of diosgenin on autophagy of human osteosarcoma cells.METHODS Human osteosarcoma MG63 and U2OS cells with or without exposure to diosgenin had their proliferation detected by MTT assay,their ultrastructure observed by transmission electron microscopy,their expression of autophagy protein Beclin1 observed by immunofluorescence staining,and their expressions of autophagy molecular markers LC3,Beclin1 and PI3K/Akt/mTOR signaling pathway related proteins detected by Western blot.The MG63 and U2OS cells cotreated with diosgenin and PI3K pathway inhibitor LY294002 had the expression of Beclin1 mRNA detected by RT-qPCR.The MG63 and U2OS cells cotreated with autophagy inhibitor 3-methyladenine(3-MA)had their inhibition rate of proliferation detected by MTT assay,their expression of cleaved-caspase3 protein detected by Western blot,and their expression of caspase3 mRNA detected by RT-qPCR.RESULTS Upon osteosarcoma MG63 and U2OS cells,diosgenin inhibited their proliferation,promoted the generation of autophagosomes,increased the protein expression of LC3 Ⅱ and Beclin1(P<0.05,P<0.01),reduced the protein expression of LC3 I(P<0.01),and inhibited the protein phosphorylation level of PI3K/Akt/mTOR pathway(P<0.05,P<0.01),whose effects were offset by the intervention with autophagy inhibitors in terms of the reduced proliferation inhibition and down-regulated expressions of caspase3 mRNA and cleaved-caspase3 protein(P<0.01).CONCLUSION Diosgenin can inhibit the proliferation of osteosarcoma cells and induce their autophagy leading to their death and autophagy apoptosis,which may be related to the activation of PI3K/Akt/mTOR signaling pathway and up-regulation of the expression of LC3 Ⅱ and Beclin1 proteins.

Effects of warm acupuncture on apoptosis of chondrocytes and MiR-27a-mediated PI3K/AKT/mTOR signaling pathway in a rat model of knee osteoarthritis / 中华物理医学与康复杂志

Objective:To observe any effect of warm acupuncture on chondrocyte apoptosis and the miR-27a-mediated PI3K/AKT/mTOR signaling pathway using a rat model of early knee osteoarthritis (KOA).Methods:Fifty Sprague-Dawley rats were randomly divided into a control group, a model group, a warm acupuncture group, an inhibitor group, and an inhibitor + warm acupuncture group (the combined group), each of 10. Three days before the modeling, both the inhibitor and combined groups were injected with miR-27a inhibitor. Papain was then injected in all groups except the control group to establish the early KOA model. After successful modeling, the combined and warm acupuncture groups were given 30 minutes of warm acupuncture at the medial and lateral Xiyan points daily for 14 days. The model and inhibitor groups were fixed for 30 minutes during those sessions. After the 2 weeks, hematoxylin-eosin staining was used to observe any pathological changes in the cartilage tissue. Terminal deoxynucleoitidyl transferase-mediated nick end labeling was used to detect chondrocyte apoptosis, and enzyme-linked immunosorbent assays were employed to observe the levels of interleukin 1β (IL-1β) and IL-6. Western blotting was used to evaluate the expression of p-PI3K, p-AKT, p-mTOR, PI3K, AKT, mTOR, LC3-II, and Beclin1 proteins in the cartilage tissue, while quantitative real-time polymerase chain reactions detected the content of miR-27a.Results:After the intervention, the morphology of the chondrocytes in the warm acupuncture group had improved significantly compared to the model group, while that of the inhibitor and combined groups was better than among the warm acupuncture group. The rate of chondrocyte apoptosis in the warm acupuncture group was significantly lower than in the model group, while the rates of the inhibitor and combined groups were lower still. There was no significant difference between the inhibitor and the combination group on average. The average expression of IL-6, IL-1β, LC3-II and Beclin1 protein and of miR-27a were significantly lower in the warm acupuncture, inhibitor and combined groups than among the model group, with those of the inhibitor and combined groups significantly lower than among the warm acupuncture group, on average. The average p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR levels of the warm acupuncture, inhibitor and combined groups were significantly higher than those of the model group, with those of the inhibitor and combined groups significantly higher, on average, than among the warm acupuncture group. However, there was no significant difference between the inhibitor group and the combined group in their protein expression and mRNA levels.Conclusions:Warm acupuncture may down-regulate the expression of miR-27a to promote the activation of the PI3K/AKT/mTOR signaling pathway, inhibiting excessive autophagy and apoptosis. That would relieve inflammation and damage, and delay degeneration in early KOA, at least in rats.

Effect of Atractylenolide Ⅰ on Lung Injury in Rats with Recurrent Respiratory Tract Infection of Lung and Spleen Qi Deficiency by Regulating the PI3K/Akt/mTOR Signaling Pathway / 中药新药与临床药理

Objective To investigate the effect of atractylenolideⅠon lung injury in rats with recurrent respiratory tract infection(RRTI)of lung and spleen qi deficiency by regulating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway.Methods Eighty-four rats were randomly separated into a control group,a model group,a low-dose atractylenolideⅠgroup,a high-dose atractylenolideⅠgroup,a positive drug group,an insulin-like growth factor-1(IGF-1)group,and a high-dose atractylenolide Ⅰ+IGF-1 group,with 12 rats in each group.Except for the control group,the RRTI rat model of lung and spleen qi deficiency was constructed using a combination of fatigue,dietary disorders,and fumigation method with shavings and tobacco among rats in other groups.After the model is successfully copied,the model was administered once a day for 6 weeks.Animal lung function instrument was applied to detect the changes of peak expiratory flow(PEF),forced expiratory volume in first second(FEV1),forced vital capacity(FVC)in rats.The changes of wet/dry mass ratio of lungs in rats were detected.HE staining was applied to detect pathological changes of lung tissue in rats of each group.ELISA was applied to detect the levels of interleukin(IL)-6,tumor necrosis factor-α(TNF-α),malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in rat lung tissue.Western Blot was applied to determine the protein expressions of p-PI3K,p-Akt,and p-mTOR in rat lung tissue.Results Compared with the control group,rats in the model group showed a decrease in PEF,FEV1 and FVC(P＜0.01)and an increase in the wet/dry mass ratio of lungs(P＜0.01).The alveolar septa in lung tissues had become larger.Pulmonary interstitial edema and a large amount of inflammatory cell infiltration were found.The levels of IL-6,TNF-α and MDA in lung tissue increased(P＜0.01),and the SOD activity decreased(P＜0.01).The protein expressions of p-PI3K,p-Akt,and p-mTOR in lung tissue increased(P＜0.01).Compared with the model group,rats in the low-,high-dose atractylenolideⅠgroups,and positive drug group showed an increase in PEF,FEV1,and FVC,and a decrease in the wet/dry mass ratio of lungs(P＜0.01).Pathologic damage in lung tissue was alleviated.The levels of IL-6,TNF-α,MDA decreased and SOD activity in lung tissue increased(P＜0.01).The protein expressions of p-PI3K,p-Akt,and p-mTOR in lung tissue decreased(P＜0.01),while the corresponding indicators in the IGF-1 group showed opposite trends(P＜0.01).Compared with the high-dose group of atractylenolide I,rats in the high-dose atractylenolide I+IGF-1 group showed a decrease in PEF,FEV1 and FVC,and an increase in the wet/dry mass ratio of lungs(P＜0.01).Pathologic damage in lung tissue was increased.The levels of IL-6,TNF-α,MDA increased and the SOD activity in lung tissue decreased(P＜0.01).The protein expressions of p-PI3K,p-Akt,and p-mTOR in lung tissue increased(P＜0.05,P＜0.01).Conclusion AtractylenolideⅠmay improve lung injury in RRTI rats of lung and spleen qi deficiency by inhibiting the PI3K/Akt/mTOR pathway.

Study on the mechanism of Compound lizard powder reducing cisplatin resistance in gastric cancer by regulating glycolysis / 中国药房

OBJECTIVE To explore the mechanism of Compound lizard powder reducing cisplatin resistance in gastric cancer by regulating glycolytic activity based on phosphoinositide 3-kinase（PI3K）/protein kinase B（Akt） signaling pathway. METHODS Human gastric cancer MKN45 and MKN45/DDP （cisplatin-resistant） cells were cultured in vitro and intervened with different mass concentrations of cisplatin （0.1， 0.2， 0.4， 0.8， 1.6， 3.2 μg/mL） to detect the survival rate， half inhibitory concentration （IC50） and drug resistance index. MKN45/DDP cells were inoculated subcutaneously in the right anterior axilla of nude mice to prepare a transplanted tumor model of gastric cancer. After successful modeling， they were randomly divided into model group， cisplatin group （0.002 g/kg）， Compound lizard powder group （2.8 g/kg） and combination group （the same dose as each single drug group）， with 8 nude mice in each group. Each administration group was given relevant solution， twice a week （cisplatin， i.p.） or twice a day （Compound lizard powder， i. g.）， for 4 consecutive weeks. During the experiment， the body weight of nude mice was monitored， and tumor volume and inhibitory rate of tumor were calculated. The levels of inflammatory factors （tumor necrosis factor- α， interleukin-6） in tumor tissue， the mRNA and protein expressions of multidrug resistance-associated protein 1 （MRP1）， P-glycoprotein （P-gp）， glucose transporter-1 （GLUT1） and lactate dehydrogenase A （LDHA）， as well as the protein expressions of PI3K， phosphorylated PI3K （p-PI3K）， Akt， phosphorylated Akt （p-Akt）， hexokinase-2 （HK2） and pyruvate kinase M2 （PKM2） were all detected. RESULTS With the intervention of different concentrations of cisplatin， the survival rate of MKN45/DDP-resistant cells was significantly higher than that of MKN45 parent cells （P＜0.05）. IC50 value of MKN45/DDP and MKN45 cells were（1.052 0±0.221 9） and （0.372 1±0.238 0）μg/mL， and the drug resistant index was 2.827. Compared with the model group， cisplatin group， Compound lizard powder group and combination group all had certain inhibitory effects on the tumor growth in nude mice； the inhibitory rates of tumor increased significantly （P＜0.05）； the levels of inflammatory factors， the mRNA and protein expressions of MRP1， P-gp， GLUT1 and LDHA （except for cisplatin group）， the phosphorylation levels of PI3K and Akt protein （except for cisplatin group） as well as the protein expressions of HK2 and PKM2 were decreased significantly， while the combination group was significantly better than the cisplatin group （P＜0.05）. CONCLUSIONS Compound lizard powder may inhibit tumor growth in transplanted tumor model nude mice with gastric cancer-resistant cells by reducing the secretion of tumor-related inflammatory factors， inhibiting the expression of glycolysis， drug resistance-related proteins and genes， inhibiting the activation of the PI3K/Akt signaling pathway， thus having a certain effect of enhancing cisplatin efficacy and reversing drug resistance.

Effect of Irisin on islet β cell function in type 2 diabetic rats by regulating autophagy through PI3K/AKT/mTOR pathway / 中国生物制品学杂志

@#Objective To investigate whether Irisin improves islet β cells function in rats with type 2 diabetes mellitus(T2DM)by enhancing autophagy through the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway,so as to provide new ideas for clinical treatment of chronic metabolic diseases such as T2DM and metabolic syndrome.Methods Thirty SD male rats were randomly divided into normal group(NC group),T2DM group,and Irisin intervention group(T2DM + Irisin group). High-fat and high-sugar diet for 5 weeks plus low-dose(35 mg/kg)streptozotocin(STZ)induced T2DM rat model. After 8 weeks of intraperitoneal injection of Irisin,rats were tested for fasting blood glucose(FBG),fasting insulin(FINS),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C). The intraperitoneal glucose tolerance test(IPGTT),intraperitoneal insulin tolerance test(IPITT)and hyperglycemic clamp test were performed to assess the islet function and insulin resistance level of rats in each group. The expression levels of PI3K/AKT/mTOR pathway proteins and autophagyrelated proteins in the pancreas were subsequently detected by Western blot. The expression levels of insulin,microtubuleassociated protein 1 light chain 3(MAP1LC3),p62,and lysosomal associated membrane protein-2(LAMP-2)in rat pancreas were detected by immunohistochemistry(IHC).Results There was an interaction between FBG and intervention time in rats(F = 11. 751,P = 0. 000),and the FBG gradually decreased in the T2DM + Irisin group with the prolongation of the intervention time. From the 4th week of intervention,the FBG in the T2DM + Irisin group decreased significantly compared with that in the T2DM group(F = 1 008. 870,P = 0. 000). Compared with NC group,the serum concentrations of TC,TG,and LDL-C concentrations in the T2DM group significantly increased(each P = 0. 000),while the HDL-C concentrations significantly decreased(P = 0. 000). After Irisin intervention,the above indexes were all improved(P = 0. 010,0. 000,0. 000 and 0. 000,respectively). Western blot results showed that compared with NC group,p62 protein expression and LC3Ⅱ/LC3Ⅰincreased significantly(P = 0. 008 and 0. 048,respectively),and LAMP-2 protein expression decreased significantly(P = 0. 000)in T2DM group. LC3Ⅱ/LC3Ⅰexpression level further increased after Irisin intervention(P =0. 000),but p62 protein level significantly decreased(P = 0. 047)and LAMP-2 protein expression increased significantly(P = 0. 000),and the IHC results were consistent with the Western blot results. The levels of p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR decreased significantly in the T2DM group compared with NC group(P = 0. 006,0. 031 and0. 000,respectively),and the above indicators further decreased after Irisin intervention(P = 0. 033,0. 013 and 0. 000,respectively).Conclusion Irisin can enhance autophagy through PI3K/AKT/mTOR signaling pathway to improve islet βcells function,providing a new idea for the treatment of T2DM.