| Objective:Cellular and Molecular Biology-Based Technologies,mouse podocyte MPC5 was used to study the effects of compound serpentine capsule on the function and cytoskeleton of adriamycin-damaged podocytes.The mechanism of reducing proteinuria and protecting podocytes from protein level was investigated.Method:1.Mouse podocyte MPC5 were randomly divided into six groups: blank control group,doxorubicin-induced podocyte injury group,positive control group,compound serong Shelong high dose group,compound serong Shelong middle dose group,compound serong Shelong low dose group.2.Detection of podocyte viability in CCK-8 groups.3.The morphological changes of podocyte cytoskeleton were observed by fluorescence microscope.4.Western blotting(Western-blot)was used to detect the expression of mitotic septal protein neph1?FAT and cytoskeletal protein ?-actimin-4 in each group of podocytes.5.Apoptosis index of podocytes was detected Annexin V-FITC/PI double staining6.Western blotting(Western-blot)was used to detect the expression of mitochondrial central regulators PGC-1 ? ? anti-apoptotic factors Bcl-2 ?anti-apoptotic factor Bax and apoptosis-related factor caspase3?caspase7?caspase8?caspase9?caspase12 proteins in podocytes of each group.Results:1.The results of CCK-8 detection of podocyte viability in each group indicated that the cell viability in the model group was extremely significantly lower than that in the normal group(P<0.001),and the model replication was successful.comparedwith the model group,the cell viability in the middle and high dose group of Compound Shelong Capsule was significantly improved(P<0.05,P<0.01,).2.Observation of the morphology of the podocyte cytoskeleton showed that the cytofilament skeleton was red and the nucleus was blue.Compared with the normal group,the red staining was light and dark,the damage was obvious,the number of cells was decreased and the distribution was uneven,the network was fuzzy and sparse,and there were many breaks.Compared with the model group,the red staining of the cytosolic microfilament skeleton in the positive group and the compound Shelong in each dose group was deepened,and the number of cells was more and the distribution was uniform The cell microfilaments network was clear and the microfilaments were less broken.The high dose group of Compound Shelong Capsule had the same effect as the positive control group.3.Results of Western blotting(Western-blot)showed that significant decrease in protein expression(P<0.01)significantly decreased(P<0.01),the expression of ?-actinin-4 protein was significantly increased(P<0.001),and the replication of doxorubicin-damaged podocyte model was successful.Compared with the model group,the expression of neph1 ? FAT protein in the positive control group was significantly decreased(P<0.05),and the expression of ?-actinin-4 protein in the high dose group was significantly decreased(P<0.001).The expression of ?-actinin-4 protein in the high dose group of Compound Shelong Capsule decreased significantly(P<0.01)compared with the positive control group,and the expression of?-actinin-4 protein in the middle dose group of Compound Shelong Capsule decreased(P<0.05).4.Annexin V-FITC/PI results of quantitative determination of podocyte apoptosis in each group by double staining showed that the apoptosis rate of the model group was significantly increased(P<0.001)compared with that of the normal group,and the model of doxorubicin-damaged podocytes was successfully replicated;compared with the model group,the apoptosis rate of compound serong Shelong in middle and high dose group and positive control group was significantly decreased(P<0.001),and that of compound serong Shelong in low dose group(P<0.01).5.The results of Western blotting(Western-blot)showed that the expression of PGC-1??Bcl-2 protein in the model group was significantly decreased(P<0.001),the expression of 53 k D and 34 k D?caspase9-35 k D?caspase12 proteins in the Bax?caspase3-17 k D?caspase7?caspase8 group was significantly increased(P<0.001)and the expression of caspase8-57 k D protein was increased(P<0.05)compared with thatin the normal group.The expression of PGC-1??Bcl-2 protein was significantly increased(P<0.001),the expression of Bax?caspase7?caspase8-53 k D?caspase9-35 k D protein was significantly decreased(P<0.001),the expression of caspase3-17 k D ?caspase8-34 k D ? caspase12 protein was significantly decreased(#P<0.01),and the expression of casebase8-57 k D protein was decreased(P<0.05).The protein expression caspase7 ? caspase9-35 k D the high dose group of Long Shelong was extremely significantly decreased(P<0.001);the expression of Bcl-2 protein in the middle dose group of Compound Shelong Capsule was increased(P<0.05);the expression of caspase8-53 k D protein in the low dose group of Compound Shelong Capsule was extremely significantly decreased(P<0.001),and the expression of caspase9-35 k D protein was decreased(P<0.05).Conclusion:1.Compound Shelong Capsule can effectively increase the cell viability of doxorubicin injured podocytes and reduce its apoptosis rate.2.Compound Shelong Capsule protects the cytoskeleton of doxorubicin-damaged podocytes.3.Compound Shelong Capsule may protect the normal function of podocytes by up-regulating the expression of mitotic membrane protein neph1?FAT with podocyte protection,down-regulating the expression of actin cytoskeleton ?-actinin-4 protein,and repairing the integrity of mitotic membrane and cytoskeleton.4.Compound Shelong Capsule may delay apoptosis and protect the normal function of podocytes by upregulating mitochondrial central regulator PGC-1 ? ?anti-apoptotic factor Bcl-2,downregulating the expression of apoptosis-promoting factor caspase3?caspase7? apoptosis-initiating factor caspase8?9,inflammatory expression. |
PDF Full Text Request