| Chronic glomerulonephritis is the primary cause of end-stage renal disease in China.Proteinuria is one of the main clinical manifestations of chronic glomerulonephritis and the core markers of disease progression.Podocyte actin cytoskeleton rearrangement can increase podocyte activity,causing podocytes fusion and thus damage podocytes,which is one of the important mechanisms leading to proteinuria.Traditional Chinese medicine has a comprehensive effect in reducing proteinuria and delaying the progress of renal function.On the basis of years of clinical experience of renowned nephropathy expert Professor Dai Xiwen,we summarized the experience of treating chronic nephritis by YiQiQingReGao Formula,with dramatic anti-proteinuria effects.But the mechanism remains unclear.Based on previous studies,this study aims to clarify whether YiQiQingReGao Formula can reduce podocyte injury by influencing the rearrangement of podocyte actin cytoskeleton,thereby reducing proteinuria,and to explore its mechanism.This study was carried out from two aspects in bothin vivo and in vitro experiments:(1)In vivo studies were conducted to observe the effects of YiQiQingReGao Formula on podocyte injury and podocyte cytoskeleton regulation related proteins by establishing puromycin aminonucleoside nephropathy rat model.(2)In virtostudies were conducted to observe the effect of YiQiQingReGao Formula on the rearrangement of podocyte cytoskeleton induced by PAN injury,and to detect the expression of key molecules of RhoA/ROCK signaling pathway and phosphorylation level of important downstream targets,and to further explore the effect of YiQiQingReGao Formula on RhoA/ROCK signaling pathway.Part 1 Study on the mechanism of YiQiQingReGao Formula in alleviating podocyte injury in puromycin aminonucleoside nephropathy ratObjective:To observe the intervention effect of YiQiQingReGao Formula on puromycin aminonucleoside nephropathy rat,and to explore its possible mechanism from the expression of actin skeleton-related protein.Methods:40 male Wistar rats were randomly divided into sham group,model group,YiQiQingReGao Formula group and cyclosporine A group.PAN injury model was established by single intrajugular injection of PAN(40 mg/kg body weight).After the model was established,the rats in YiQiQingReGao Formula group were given 4 g/kg body weight,cyclosporine A group was given 21 mg/kg body weight,sham group and model group were given distilled water of equal volume.24-hour urinary protein were measured on the 5th and 10th days respectively,andserum biochemical parameters were measured on the 11th day.The pathological and ultrastructural changes of kidney were observed.The quantitative expression of actin cytoskeleton regulatory proteins(alpha-actinin-4,Synaptopodin,Desmin,Nestin,Vimentin and uPAR)and the expression of Rho-related GTP binding protease in kidney tissue were detected by RT-PCR,Western Blot,and ELISA accordingly.Result:(1)Clinical Parameters:Compared with the sham group,the urinary protein content in the other groups increased significantly on the 5th day after model establishment(P<0.01);compared with the model group,the YiQiQingReGao Formula group and cyclosporine A group could significantly reduce the urinary protein content in the PAN rat model(P<0.05),but there was no significant difference between the two groups(P>0.05).On the 10th day after the establishment of the model,the urinary protein content in YiQiQingReGao Formula group and cyclosporine A group was significantly lower than that in the model group(P<0.05).The levels of BUN,SCr,TC,TG and LDL in the model group were significantly higher than those in the sham group,and the levels of ALB in the model group were significantly lower than those in the sham group(P<0.05).The levels of BUN,SCr,and TG in YiQiQingReGao Formula group were significantly decreased and ALB level was increased(P<0.05),and the effect of YiQiQingReGao Formula was better than that of cyclosporine A(P<0.05)(2)Pathological changes:Light microscopy showed the number of mesangial cells increased slightly,mesangial matrix proliferated,some balloons adhered in the model group;renal tubular epithelial cells showed slight vacuolar degeneration,and a small amount of inflammatory cells infiltrated in the renal interstitium.The proliferation of mesangial cells and stroma in YiQiQingReGao Formula group was not obvious,the epithelial cells of renal tubules were slightly vacuolated,and there was no infiltration of inflammatory cells in renal interstitium.The results of transmission electron microscopy showed that in the model group,the foot processes fused extensively,the foot process Width increased significantly(P<0.05),the gap between the fissures disappeared,the endothelial window holes were disordered,the mitochondria in the podocytes swelled,the mesangial cell proliferated,and the basement membrane area had no obvious pathological changes.Compared with the model group,in the YiQiQingReGao Formula group the fusion of foot process was significantly improved,the width of foot process was significantly reduced(P<0.05),some of the foot processes returned to normal,and the columnar foot processes were arranged on the basement membrane side.(3)RT-PCR:The levels of alpha-actinin-4,Synaptopodin,Nestin,Desmin,Vimentin and uPAR in the model group were significantly higher than those in the sham group(P<0.01).Compared with model group,the expression of alpha-actinin-4,Synaptopodin and Nestin in kidney tissue was significantly decreased in YiQiQingReGao Formula group(P<0.01),and the expression of Desmin and uPAR was also down-regulated(P<0.05).The expression of Vimentin in YiQiQingReGao Formula group was not significantly different from that in model group(P>0.05).The effect of YiQiQingReGao Formula group on Synaptopodin and Nestin was similar to that of cyclosporine A group(P>0.05).(4)Western Blot:The protein expression levels of alpha-actinin-4,Synaptopodin,Desmin,Nestin,Vimentin and uPAR in the model group were significantly higher than those in the sham group(P<0.01).Compared with the model group,the expression of alpha-actinin-4 protein,Synaptopodin protein and Nestin protein in kidney tissue of YiQiQingReGao Formula group decreased significantly(P<0.01),the expression of Desmin protein and uPAR protein decreased significantly(P<0.05),and the expression of Vimentin protein had no significant difference(P>0.05).The down-regulation of Nestin expression in YiQiQingReGao Formula group was similar to that in CsA group(P>0.05).(5)ELISA:The expression of Rho-related GTP-binding protease in model group was significantly higher than that in sham group(P<0.01);compared with model group,YiQiQingReGao Formula could reduce the expression of Rho-GTPase(P<0.05).The expression level of Rho GTPase in CsA group was lower than that in YiQiQingReGao Formula group(P>0.05).Conclusion:YiQiQingReGao Formula can effectively reduce 24-hour urinary protein content and improve renal function in PAN rats.It can alleviate glomerular pathological damage,improve foot process fusion,repair podocyte injury and reduce actin cytoskeleton reorganization induced by PAN.Its mechanism may be related to down-regulation of actin cytoskeleton-related regulatory protein expression.Part 2 Repairing effects of podocyte cytoskeleton rearrangement induced by purinomycin aminonucleoside with YiQiQingReGao FormulaObjective:To observe the intervention effect of YiQiQingReGao Formula-containing serum on PAN-induced podocyte injury model.Methods:Podocyte injury model induced by PAN stimulation was established in immortal mouse podocyte lines under in vitro culture conditions.CCK-8 method,LDH release test and immunofluorescence method were used to determine the optimal concentration and intervening time(100 ug/ml intervening for 24 hours).The YiQiQingReGao Formula-containing serum and cyclosporine was prepared and divided into control group,PAN group,low-dose group(YQ-L),middle-dose group(YQ-M),high-dose group(YQ-H)and cyclosporine A group(CsA)to interfere with podocytes respectively.The effects of YiQiQingReGao Formula on podocyte viability,podocyte injury,podocyte proliferation and apoptosis,morphological changes of organelles,podocyte activity,actin skeleton rearrangement,and expression of cytoskeleton protein were detected.Result:(1)Podocyte Injury:Compared with the control group,the podocyte viability and LDH release rate of PAN group decreased significantly(P<0.01);compared with PAN group,the podocyte viability of YQ-L group increased,but the LDH release rate did not change significantly(P>0.05);the podocyte viability of YQ-M group,YQ-H group and CsA group increased significantly,and the LDH release rate decreased significantly(P<0.01).The results of transmission electron microscopy(TEM)showed that there were a lot of lipid droplets deposited in the cells of PAN group compared with those of Control group.Lots of vesicles of endoplasmic reticulum were dilated and mitochondrial cristae was disordered.Compared with PAN group,the damage of podocytes in YQ-M group,YQ-H group and CsA group was reduced,and the normal structure of mitochondria and endoplasmic reticulum was restored.(2)Cell cycle and cell apoptosis:Compared with the control group,the number of G0-G1 phase cells increased and the number of S phase cells decreased in PAN group(P<0.01).Compared with PAN group,the number of G0-G1 phase cells decreased and the number of S phase cells increased(P<0.01);CsA group had no significant effect on G0-G1 phase cells,but the number of S phase cells increased(P<0.01).The apoptotic rate of podocytes in PAN group was significantly higher than that in control group(P<0.01).Compared with PAN group,the apoptotic rate of podocytes in YQ-M group,YQ-H group and CsA group was significantly lowered(P<0.01).(3)Podocyte Mobility:Compared with the control group,the migration distance and the number of migrating cells of podocytes in PAN group increased significantly and the number of adherent cells decreased significantly(P<0.01);compared with PAN group,the migration distance of podocytes in YQ-M group,YQ-H group and CsA group decreased significantly,and the number of migrating cells decreased significantly(P<0.01).YQ-M group and YQ-H group also increased the number of adhesion cells(P<0.01).(4)Immunofluorescence staining:The body size of podocytes in PAN group was smaller,there were residual cut-off filamentous structures in the cells,the stress fibers were reduced,a lot of small protrusions and spines were protruded around the disordered cells,and the expression of Synaptopodin increased around the nucleus.In YQ-M group and YQ-H group,the damage mentioned above was alleviated.The cytoskeleton morphology of YQ-H group was better than that of YQ-M group.In YQ-M group,there were still a few spines and contractile rings formed by actin microfilaments.Synaptopodin was still highly expressed around the nucleus in YQ-M group.Synaptopodin was evenly distributed in YQ-H group and granular around the plate pseudopodia formed around the cells.Compared with YQ-M group and YQ-H group,CsA group had poorer cytoskeleton morphology,smaller cell body,less stress fibers,and no obvious polar distribution.A large number of short actin bundles were found around the nucleus,while Synaptopodin was concentrated around the nucleus.(5)RT-PCR:The levels of alpha-actinin-4,Synaptopodin,Nestin,Desmin,Vimentin and uPAR in PAN group were significantly higher than those in Control group,while the levels of Nephrin and Podocin were significantly lower(P<0.01).Compared with PAN group,YQ-H group could significantly down-regulate the expression of alpha-actinin-4,Synaptopodin,Nestin,Desmin and uPAR in podocytes,and up-regulate the expression of Nephrin and Podocin(P<0.01).YQ-M group and YQ-H group had no significant effect on Vimentin expression(P>0.05).(6)Western Blot:The protein expression levels of cytoskeleton proteins alpha-actinin-4,Synaptopodin,Desmin,Nestin,Vimentin and uPAR in PAN group were significantly higher than those in Control group,while the protein expression levels of SD proteins Nephrin and Podocin were significantly lower(P<0.01).Compared with PAN group,YQ-H group could significantly down-regulate the protein expression of Synaptopodin,Nestin and uPAR in podocytes,up-regulate the protein expression of Nephrin(P<0.01),down-regulate the protein expression levels of alpha-actinin-4,Desmin and Vimentin,and up-regulate the protein expression of Podocin(P<0.05).YQ-M group could down-regulate the expression of uPAR(P<0.01),but had no significant effect on the expression of other proteins(P>0.05).In CsA group,the protein expression levels of podocyte alpha-actinin-4,Synaptopodin,Desmin,Nestin,Vimentin and uPAR were significantly down-regulated,and the protein expression levels of Nephrin and P odocin were up-regulated(P<0.05).Conclusions:YiQiQingReGao Formula can alleviate PAN-induced podocyte injury,promote podocyte proliferation and inhibit apoptosis;it can also reduce the ability of podocyte movement and increase its adhesion ability;YiQiQingReGao Formula can inhibit actin filament rearrangement,stabilize the cytoskeleton,and restore abnormal expression of cytoskeleton actin and hiatal membrane protein.Part 3:The effect of YiQiQingReGao Formula on RhoA/ROCK signaling pathway based on podocyte actin cytoskeleton rearrangementObjective:To observe whether YiQiQingReGao Formula inhibits the rearrangement of cytoskeleton through RhoA/ROCK signaling pathway.Methods:In vitro cultured immortal mouse podocyte lines,the expression of RhoA,Rac1 and Cdc42,key molecules of Rho family small GTPase,in PAN-induced podocyte injury was detected by GLISA.It was found that YiQiQingReGao Formula could significantly increase the activity of RhoA,but had no significant effect on Rac1 and Cdc42.Then RhoA/ROCK signaling pathway inhibitor C3 transferase and RhoA/ROCK signaling pathway agonist U46619 were used to detect podocyte migration ability,cell cycle change,cytoskeleton rearrangement,and RhoA and Rho-associated coil Containing Protein Kinase 1(ROCK1)expression level,and downstream key molecule:Phosphorylation levels of myosin light chain(MLC),myosin phosphatase-targeting subunit 1(MYPT1),LIM-Kinase(LIMK),cofilin and relative expression levels of globular actin monomer(G-actin)and filamentous actin fibers(F-actin),Result:(1)GLISA:That the activities of RhoA,Racl and Cdc42 in PAN group were lower than those in Control group(P<0.01);compared with PAN group,the activity of RhoA in YQQRG group increased significantly(P<0.01),but had no significant effect on the activities of Racl and Cdc42(P>0.05).Compared with YQ group,the activity of RhoA decreased after pretreatment altogether with C3 transferase,a RhoA/ROCK signaling pathway inhibitor,and the activity of RhoA increased slightly after pretreatment with U46619,a RhoA/ROCK signaling pathway agonist(P>0.05).(2)Cell cycle:Compared with PAN group,the number of G0-G1 phase cells decreased and S phase cells increased in YQ group(P<0.05).Compared with YQ group,cells in G0-G1 phase increased and S phase decreased after pretreatment with RhoA/ROCK signaling pathway inhibitor C3 transferase(P<0.05);cells in G0-G1 phase continued to decrease after pretreatment with RhoA/ROCK signaling pathway agonist U46619(P<0.05),while cells in S phase increased slightly(P>0.05).(3)Podocyte Mobility:Compared with the control group,the number of migrating podocytes in PAN group increased significantly(P<0.01),while the number of migrating podocytes in YQ group decreased significantly(P<0.01).Compared with YQ group,the number of migrating cells increased after pretreatment with RhoA/ROCK signaling pathway inhibitor C3 transferase(P<0.05),and decreased after pretreatment with RhoA/ROCK signaling pathway agonist U46619(P>0.05).(4)Immunofluorescence staining:The cell body size of PAN group were significantly reduced,the stress fibers were decreased,the arrangement was disordered,the structure was unclear,and many small protrusions and spines were protruded around the cells;treatment of YiQiQingReGao Formula could improve the changes of the cytoskeleton structure and restore the normal arrangement of the stress fibers;Ater YiQiQingReGao Formula and RhoA/ROCK signaling pathway inhibitor C3-transferase jointly prefabricated,the cytoskeleton morphology was poorer,and a large number of short actin bundles were distributed around the cell membrane.After pretreatment with YiQiQingReGao Formula and RhoA/ROCK signaling pathway agonist U46619,the cytoskeleton morphology was better than that of YiQiQingReGao Formula alone,and there were plate-shaped pseudopods around the cells.(5)Western Blot:The expression levels of RhoA and ROCK1 in PAN group were significantly lower than those in Control group(P<0.01).Compared with PAN group,the expression levels of RhoA and ROCK1 in PAN group were elevated by YiQiQingReGao Formula(P<0.01).Compared with the pre-intervention of YiQiQingReGao Formula alone,the expression levels of RhoA and ROCK1 decreased after pretreatment with RhoA/ROCK signaling pathway inhibitor C3 transferase(P<0.01,P<0.05);after pretreatment with YiQiQingReGao Formula and RhoA/ROCK signaling pathway agonist U46619,the expression levels of RhoA and ROCK1 increased(P<0.01,P<0.05).Compared with the control group,the phosphorylation level of MYPT1 and MLC decreased,the phosphorylation level of LIMK and Cofilin also decreased,and the ratio of G-actin/F-actin increased significantly(P<0.01).Compared with the PAN group,the phosphorylation level of MYPT1 and MLC increased,and the phosphorylation level of LIMK and Cofilin also increased,and the ratio of G-actin/F-actin decreased significantly(P<0.01).Compared with the intervention of YiQiQingReGao Formula alone,C3-transferase,an inhibitor of RhoA/ROCK signaling pathway,could antagonize the increase of phosphorylation level and the decrease of G-actin/F-actin ratio(P<0.01,P<0.05);With the help of U46619,an activator of RhoA/ROCK signaling pathway,the intervention effect of YiQiQingReGao Formula was more obvious(P<0.01,P<0.05).Conclusion:YiQiQingReGao Formula can increase the activity of RhoA in damaged podocytes,but has no obvious effect on Rac1 and Cdc42.YiQiQingReGao Formula can promote the proliferation of podocytes,reduce the activity of podocytes and inhibit the rearrangement of cytoskeleton by activating RhoA/ROCK signaling pathway.Its mechanism may be through promoting the phosphorylation levels of MLC and MYPT1,and also promoting the phosphorylation levels of LIMK and ofilin,reducing the G-actin/F-actin ratio,stabilizing the cytoskeleton and exerting the protective effect of podocytes.Overall Conclusion:(1)YiQiQingReGao Formula can reduce proteinuria,improve renal function,and alleviate kidney pathological damage in PAN model rats,(2)In vitro,YiQiQingReGao Formula can alleviate podocyte injury induced by PAN,inhibit actin cytoskeleton rearrangement,and restore the normal distribution of actin filaments.(3)Its mechanism is to activate RhoA/ROCK signaling pathway,promote MLC and MYPT1 phosphorylation,promote LIMK and Cofilin phosphorylation,reduce G-actin/F-actin ratio,stabilize the cytoskeleton,and exerting the protective effect of podocytes. |
