| Diabetic nephropathy(DN)is serious kidney disease.It is also a systemic microvascular complication.Now,it has developed into a serious global public health problem.Studies have reported that 20% of diabetes patients for more than 10 years will develop to DN.In early stage of DN,the clinical symptoms are characterized by urinary microalbumin and thickening of the basement membrane.But it is difficult to detect and difficult to reverse.There is a lack of effective treatment strategies.Thus,early diagnosis,timely prevention,and treatment are essential.Podocytes and GMCs are inherent cells of the kidney.The changes have an important impact on the occurrence and development of DN.In the disease,abnormal proliferation of GMCs can lead to the deposition of the extracellular matrix,thickening of the glomerular basement membrane,and other pathological changes.Podocytes are important cells that regulate the filtration capacity of the glomerulus.Its structural integrity and number can maintain the normal filtration capacity of the kidney.So,it plays a key role in the integrity of the glomerulus.The physiological and pathological mechanisms of DN are very complex,involving genetic information,oxidative stress,inflammation,and abnormal glucose and lipid metabolism.In abnormal glucose metabolism,the expression and translocation of glucose transporters have grown up to be a hot topic.In the high glucose environment,the first rate-limiting step of cell metabolism is glucose uptake.Glucose is a polar and hydrophilic molecule.Thus,it requires a carrier protein to transport across the membrane into the cell.The regulation of glucose transporters is very important.Berberine(BBR)is an alkaloid extracted from the rhizomes of the natural plant Coptis Rhizoma.It has the effects of anti-oxidative stress,anti-inflammatory,and lowering blood glucose.Thus,studying the mechanism by which BBR affects glucose transport and regulates abnormal changes in kidney cells can better provide new directions and targets for the clinical treatment of DN.Objective: In vivo and vitro experiments,we explored the effects of BBR on function,morphology,glomerular ultrastructure of DN mice kidney,as well as the regulatory mechanism of abnormal changes in kidney cells.To explore the specific mechanism of the protective effect on the kidneys of DN mice.Methods: In vivo experiments used streptozotocin(STZ,50 mg/kg)to be injected continuously for five days and fed with a high-fat feed to establish a diabetic mice model.The fasting blood glucose(FBG)of mice were measured on 3 and 7 days,and mice with FBG ? 16.7 mmol/L were included in the experimental group.Then fed the high-fat diet for 10 weeks to prepare a DN model.The experiment is divided into high-fat model group(DN),BBR medium-dose group(90 mg/kg),BBR high-dose group(180 mg/kg),positive drug group(Metformin,200 mg/kg),and normal control group.The drug treatment group was given intragastric administration once a day,and the normal control group and the high-fat model group were given intragastric administration of sodium carboxymethyl cellulose as a control.The model group and the drug treatment group were still fed with high-fat feed,and the normal control group was fed with ordinary feed.After 10 weeks,to detect renal function parameters of mice,measure the blood urea nitrogen(BUN),urine creatinine(UCr)and total urine protein(TUP)of mice.Moreover,the pathological changes of the mice kidneys were observed by HE and PAS staining methods,the ultrastructural changes of the glomerular basement membrane of the mice were observed by transmission electron microscopy.Moreover,Western blot analyzed the changes of PI3K/AKT signaling.Immunohistochemistry was used to analyze the expression level of GLUT1 and GLUT4 in mice kidney.In vitro,the experiment was divided into two parts.First,GMCs were used as the research object.MTT and Ed U methods were used to detect the effects of different times and different concentrations of BBR on the abnormal proliferation of GMCs under normal medium and high glucose stimulation;flow cytometry was used to detect the abnormal distribution of BBR on the GMCs cycle.Western blot and RT-q PCR were used to detect the expression of BBR on PI3K/AKT/AS160/GLUT4 related molecules.Meantime,2-NBDG was used to detect the sugar uptake of high glucose-induced GMCs by BBR.Secondly,podocytes were used as the research object.Western blot was used to detect the expression of desmin and podocin.At the same time,flow cytometry was used to detect the apoptosis level of podocytes under high glucose stimulation at different times.Therefore,it can determine the time point of podocyte injury under the high glucose stimulation.2-NBDG method was used to observe the effect of BBR on the uptake of glucose in podocytes induced by high glucose;Western blot was used to screen the expression of GLUT subtypes in podocytes and BBR on the PI3K/AKT/m TOR and caspase8/caspase3 signaling pathway-related proteins in podocytes induced by high glucose.At the same time,flow cytometry and Hoechst staining were used to detect the effect of BBR on the apoptosis level of podocytes under high glucose.Results:Part 1: The protective effect of berberine on the kidneys of diabetic nephropathy mice1.Biochemical indicators showed that the values of BUN,UCr and TUP in the DN group were significantly higher than those in the normal control group(P < 0.001).Both the BBR(90,180 mg/kg)and metformin groups could improve this phenomenon to varies degrees(P < 0.001).HE and PAS staining shows that compared with the normal group,mice in the DN group had glomerular sclerosis,glycogen deposition,extracellular matrix accumulation and obvious thickening of the basement membrane.After giving different doses of BBR(90,180 mg/kg),it can significantly improve the pathological changes of the kidney and the thickening of glomerular basement membrane in DN mice,which is similar to metformin.2.The results of immunohistochemistry and Western blot showed that BBR(90,180 mg/kg)can significantly reduce the expression level of GLUT1 and GLUT4 in kidney of DN mice(P < 0.05),and its mechanism may be related to the PI3K/AKT signaling pathway.Part 2: Research on the molecular mechanism of berberine on the cell cycle of high glucose-induced GMCs1.MTT and trypan blue staining showed that compared with the NC group.GMCs proliferated abnormally after 24 hours of culture in high glucose(P < 0.001);the abnormality could be improved to varying degrees after BBR treatment(P < 0.05).2.Ed U results showed that with the increase of high glucose stimulation time,GMCs began to proliferate abnormally at 20 h(P < 0.05),and with the increase of time,the abnormal cell proliferation phenomenon became more significant.3.The results of flow cytometry indicated that the proliferation cycle of GMCs changed under high glucose stimulation,which significantly prevented GMCs from entering the G1 phase(P < 0.05),and accelerated G1 cells to enter the S phase(P <0.001),but had almost no significant effect on the G2 phase(P > 0.05).After BBR intervention treatment,it can not only increase the proportion of G1 phase,but also reduce the proportion of S phase,and this trend becomes more significant over time(P< 0.001).4.Western blot and RT-q PCR results showed that BBR can significantly inhibit the proteins changes of PI3 K,p-AKT,p-AS160 and m-GLUT4 induced by high glucose(P< 0.05).At the same time,BBR also inhibited the m RNA expression of PI3 K,AKT,AS160 and GLUT4(P < 0.05).5.2-NBDG method measured glucose uptake and found that high glucose significantly increased the glucose uptake of GMCs(P < 0.001),and BBR could significantly improve this phenomenon.At the same time,BBR can significantly regulate the aberrant cell cycle of GMCs.Part 3: Effect of berberine on the autophagy and apoptosis in podocytes induced by high glucose1.Western blot results showed that the protein expression of desmin was significantly up-regulated after high glucose stimulation for 12 h(P < 0.01);the protein expression level of podocin,a podocyte-specific protein,was significantly reduced at 24h(P < 0.01).2.Ed U staining showed that with the increase of high glucose stimulation time,the number of podocyte proliferation decreased.3.The results of flow cytometry showed that with the increase of high glucose stimulation time,significant apoptosis of podocytes occurred at 24 h(P < 0.01).4.Western blot results showed that the protein expression of Total-GLUT1,2,4was increased,but it was not significant(P < 0.05);the protein expression of m-GLUT1,2 significantly increased(P < 0.05).5.The results of 2-NBDG indicate that BBR can reduce the abnormality of glucose uptake under high glucose condition.6.Western blot results indicate that BBR can inhibit the expression of PI3K/AKT/m TOR and caspase8/caspase3 signaling pathway-related proteins in high glucose(P < 0.05).7.The results of flow cytometry and Hoechst staining showed that BBR can reduce podocyte apoptosis under high glucose(P < 0.05).Conclusion:1.The protective effect of BBR on the kidneys of DN mice may be related to activating the PI3K/AKT signaling pathway and improving glycogen deposition.2.In vitro studies have shown that BBR inhibits GLUT4 transship and cell cycle distribution of GMCs may be related to the PI3K/AKT/AS160/GLUT4 pathway.3.BBR can regulate the autophagy and apoptosis of podocytes under high glucose conditions through PI3K/AKT/m TOR and caspase8/caspase3 signaling pathways,and alleviate the phenomenon of reduced podocytes,which may involve glucose transport. |
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