| Objective:Chronic kidney disease(CKD)is characterized as kidney injury leaded by various causes and/or glomerular filtration rate(GFR)decline,including glomerular diseases,renal tubular interstitial diseases,renal vascular diseases and so on.CKD has become a worldwide public health problem,so it is of great medical value and social significance to carry out research on the treatment and treatment mechanism of CKD.The foot processes of podocytes and slit diaphragms are the important components of glomerular filtration barrier,podocytes could generate many components of the renin-angiotensin system(RAS)and they express receptors of RAS,including angiotensin?(Ang ?),mineralocorticoid,and prorenin receptors.Crucial functions of podocytes,including contraction,apoptosis,autophagocytosis and cytoskeletal organization,have been shown to be regulated by the Ang ? type 1 receptors.Reactive oxide species(ROS)are involved in the initiation and outcome of autophagy,and autophagy can remove damaged mitochondria and oxidative proteins,which is an important protective mechanism for cell damage caused by ROS and can alleviate cell damage.Ang ? can enhance autophagy through increasing ROS levels in podocytes,inhibitors of oxidative stress could down-regulated autophagy.Previous research results showed that Modified Huangqi Chifeng Decoction(MHCD)could inhibit the excessive autophagy induced by adriamycin and alleviate the oxidative stress injury of rats with adriamycin nephropathy.In view of the important role of RAS in renal lesions,we established the Ang ?-induced injury model of podocytes,investigating the mechanism of MHCD alleviates the injury of glomerular podocytes induced by Ang ? based on the regulation of ROS-autophagy pathway.Methods:The drug-containing serum of MHCD and telmisartan was prepared,and the toxic effect of drug-containing serum on podocytes was detected by CCK-8.Concentrations of Ang ? from 10-8mol/L to 10-4mol/L was selected to incubate podocytes for 24h,CCK-8 was adopted to detect the viability of podocytes;concentrations of Ang ? 10-8mol/L?10-7mol/L?10-6mol/L was selected to incubate podocytes for 24h,western blot was adopted to detect the expressions of nephrin and podocin.Based on the results of CCK-8 and western blot,establish the Ang ? induced injury model of podocytes.To research the effect of MHCD on the injury and autophagy of podocytes,detecting the expressions of nephrin,podocin,LC3,Beclin-1 and p62 by western blot,detecting the activity of autophagy by lysosomes fluorescent probe,and observing the formation of autophagosome by transmission electron microscope(TEM),intracellular reactive oxide species(ROS)levels were determined by flow cytometer.To investigate the protective effect of MHCD on the podocytes by regulating the ROS-autophagy pathway.Results:1.CCK-8 method was used to detect the activity of podocytes,the results showed that the OD value of MHCD and telmisartan-containing serum group decreased slightly compared with that in blank group,but there was no statistically difference in OD value between groups(P>0.05).The results showed that the MHCD and telmisartan-containing serum had no toxic effect on podocytes.2.Establish Ang ? induced injury model of podocytes:Detect the effect of Ang? on the viability of podocytes by CCK-8,compared with the control group,the viability of the podocytes intervened by different concentrations from 10-8mol/L to 10-4mol/L Ang ? for 24h was decreased,but there was no statistically significant difference(P>0.05);the effect of Ang ? on the expressions of nephrin and podocin of injury-related proteins in podocytes was detected by western blot,10-8mol/L,10-7mol/L,10-6mol/L Ang ? lead to the decreases of nephrin and podocin,there was no significant difference between the 10-8mol/L Ang ? group and the control group(P>0.05);the differences were statistically significant between 10-7mol/L,10"6mol/L Ang ? group and the control group(P<0.05).In reference,the induction of autophagy was not stronger than that of 10-7mol/L and 10-8mol/L when the concentration of Ang ? was 10-6mol/L,and 10-8mol/L Ang ? could not induce the injury of podocytes.The purpose of this experiment was to explore the effect of MHCD on Ang ? induced injury of podocytes and autophagy flow,finally,10-7 mol/L Ang ? was chosen for Ang ?induced injury model of podocytes.3.Ang ? decreased the expressions of nephrin and podocin of podocytes,compared with the control group,the differences were statistically significant(P<0.01);MHCD and telmisartan increase the expressions of nephrin and podocin,there were no significant differences between the MHCD low dose group and the model group(P>0.05);compared with the model group,MHCD middle dose group,MHCD high dose group and telmsartan group significantly increased the expressions of nephrin and podocin(P<0.01).4.Compared with the model group,3MA and chloroquine decreased the viability of podocytes,the differences were statistically significant(P<0.05);compared with the model group,3MA and chloroquine decreased the expressions of nephrin and podocin of podocytes,the differences were statistically significant(P<0.05).5.Ang ? increased the expressions of autophagy-associated protein LC3 ?/LC3 I and Beclin-1,decreased the expression of p62,compared with the blank group,the differences were statistically significant(P<0.01);MHCD and telmisartan decreased the expressions of autophagy-associated protein LC3 ?/LC3 ? and Beclin-1,increased the expression of p62,compared with the control group(P<0.05).6.The results of TEM showed that the structure of the podocytes was intact,the nuclear membrane and nucleolus were clear,the structure of cell organelle was normal,the autophagosomes were fewer in the control group.In the model group,the number of autophagosomes were increased,cell organelles can be contained in the autophagosomes.MHCD and telmisartan decreased the number of autophagosomes.The results showed that Ang II could induce autophagy of podocytes,and the MHCD and telmisartan could inhibit the autophagy of podocytes,which was consistent with the results of western blot results.7.In the process of autophagy,the autophagy needs to be combined with the lysosomal to complete the degradation of the substrate,the lysozyme is combined with autophagy to convert the primary lysosomal into a secondary lysosomal,with an elevated acidity,which has the function of degrading content,and the lysozyme fluorescence probe can mark acidic lysosomal and reflect the function of lysosomal.After the drug-containing serum intervened in the podocytes,the changes of lysosomal were observed with fluorescence microscope.The results showed that the expression of lysozyme in model group was enhanced,and the expression of lysozyme was reduced after the addition of MHCD and telmisartan intervention.The results showed that Ang ? can not only induce the initiation of autophagy of podocytes,but also promote the combination of autophagy and lysosomal,upregulating the level of autophagy;on the contrary,the MHCD and telmisartan inhibited the level of the whole autophagy.8.The expression of ROS in podocytes was detected by flow cytometer.Compared with the control group,the expression of ROS in the model group increased(P<0.01);compared with the model group,the expression of ROS in each dosage group of MHCD and telmisartan group decreased(P<0.05).Ang ? induced the expression of ROS in podocytes,and the expression of ROS was reduced by MHCD.Conclusions:1.Ang ? induced injury of podocytes by decreasing the expressions of nephrin and podocin of podocytes.2.MHCD alleviates injury of podocytes by increasing the expressions of nephrin and podocin of podocytes.3.Autophagy induced by Ang ? has the function of protecting the podocytes,inhibition of autophagy decreases the viability of podocytes.4.Based on the regulation of ROS-autophagy pathway,MHCD may downregulates autophagy flux induced by Ang ?,alleviating the injury of glomerular podocytes. |
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