| Objective:With high glucose-induced renal podocyte injury in mice as the research object,the effects of serum containing compound Xiancao granules on nephrin and PERK-ATF4-CHOP signaling pathways of renal podocyte marker proteins were studied.The protective mechanism of Zhuang medicine compound Xiancao granules on high glucose-induced renal podocyte injury was expounded at the molecular level,so as to further reveal the mechanism of this prescription on diabetic nephropathy(DN).Methods:(1)A total of 100 8-week-old SD rats were randomly divided into the high,medium and low dose groups of Compound Xiancao Granules(20rats in each group)and the pure water control group(40 rats in each group),once a day.After continuous intragastric administration for 7 days,rats were anesthetized with pentobarbital sodium,and the serum of drug-containing rats and blank rats were prepared by blood collection from the abdominal aorta.(2)The mouse renal podocytes were divided into blank control group(fetal bovine serum control group),negative control group(blank rat serum control group),model control group,metformin control group,irbesartan control group,different concentrations of compound Xiancao granule medicated serum group,respectively,to intervene in vitro culture 24 h,48h,72 h.CCK-8method was used to determine the effect of compound Xiancao granules on the proliferation activity of renal podocyte cells.The apoptosis rate of middle and high concentration group was significantly lower than that of model group(P< 0.05).(3)Flow cytometry was used to determine the apoptosis rate of renal podocytes;(4)Transmission electron microscopy for morphological observation of renal podocytes;(5)Western blot to detect the expression of apoptotic proteins;(6)The m RNA expression of the target gene was detected by RT-PCR.Results:(1)CCK-8 method was used to determine the effect of compound Xiancao granules on the proliferation activity of renal podocytes.The results showed that the renal podocyte injury was obvious in the model group,and the serum containing compound Xiancao granules at different concentrations had protective effects on renal podocyte injury(P<0.05),But the dose dependence was not obvious(P>0.05);(2)The apoptosis rate of renal podocytes measured by flow cytometry showed that there were differences in the apoptosis rate of each group,and the apoptosis rate in the model group was the highest(P<0.05),the blank control group and negative control group were similar(P>0.05),which proved that blank rat serum had no toxic damage to mouse renal podocytes.The apoptosis rate of irbesartan group was lower than that of model group and metformin group(P<0.05),the metformin control group was similar to the model group(P>0.05),indicating that irbesartan has better protective effect on renal podocyte injury induced by high glucose than metformin.The apoptosis rate of renal podocytes in compound Xiancao granules low concentration group was similar to that in blank group,negative group and metformin control group(P>0.05).It is proved that compound Xiancao granule has protective effect on renal podocyte injury induced by high glucose and does not depend on hypoglycemic.The apoptosis rate of different concentrations of compound Xiancao granule medicated serum groups was different with the change of concentration.The apoptosis rate decreased with the increase of low,medium and high concentrations(P<0.05),and there was a dose-dependent trend.(3)The morphology of renal podocytes observed by electron microscope showed that there were certain differences in each group.The cell damage of model control group was the most serious,and the cell structure of each administration group was significantly restored compared with model group.The overall structure of the blank control group and the negative control group was still acceptable,which proved that the blank rat serum did not cause damage to the mouse renal podocytes.(4)There were differences in the expression levels of apoptotic proteins in each group.The expression of apoptotic proteins in the model group was the highest,and that in the drug group was decreased(P<0.05).(5)The expression of nephrin m RNA in the model group was decreased,while that in the medication group was increased(P<0.05).The m RNA expression of PERK-ATF4-CHOP signaling pathway related genes increased in model group and decreased in drug group(P<0.05).Conclusion:(1)Zhuang medicine compound Xiancao granules can promote the proliferation of damaged renal podocytes induced by high glucose and inhibit their apoptosis;(2)Zhuang medicine compound Xiancao granules can improve endoplasmic reticulum stress(ERS)in renal podocytes,which is manifested by reducing the elevated apoptotic protein level of ERS induced by high glucose,the expression levels of PERK-ATF4-CHOP pathway-related genes and apoptotic genes,and increasing the m RNA expression level of nephrin gene.The mechanism of its treatment of DN may be related to the regulation of PERK-ATF4-CHOP apoptosis signaling pathway in ERS induced by high glucose. |
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