| Objective:To further investigate the molecular mechanism study of Suoquan Yishen Formula to regulate EMT of podocyte by interfering with miR-378 d to inhibit KDM6A/KLF10 signaling axis and explain its protection of podocyte.Methods:1.in vivo study:To study the mechanism of renal protection of Suoquan Yishen formula(SQYSF)in mice with diabetic nephropathy,db/m mice were selected as a negative control group(db/m group)and spontaneous type 2 diabetic db/db mice were fed adaptively for 2 weeks and randomly divided into db/db model group(db/db group),db/db+Suoquan Yishen formula group(db/db + SQYSF group)and db/db +Canagliflozingroup(db/db + Cana group)was divided into 6 mice and gavaged for 16 weeks.Blood glucose,body weight and urine microalbumin(m Alb)were measured every 2 weeks.After 16 weeks,mices were anesthetized and executed to obtain blood and kidney tissues for the detection of blood creatinine(Scr),blood urea nitrogen(BUN),hematoxylin-eosin staining(HE),periodate Schiff reaction(PAS),Masson trichrome staining(Masson)to observe histopathological changes in the kidney,and real-time fluorescence quantitative polymerase The expression of KDM6 A,KLF10,α-SMA,N-cadherin,P-cadherin and nephrin mRNA and protein in kidney tissues were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and protein immunoblotting(Western blot),and the expression level of miR-378 d in kidney tissues was measured by RT-qPCR.2.In vitro studies:Investigating the mechanism of action of SQYSF in regulating epithelialmesenchymal transition(EMT)of podocyte through miR-378 d inhibitor of KDM6A/KLF10 signaling axis.(1)Intervention of SQYSF-containing serum on the damage of podocytes under high glucose(HG)stress and EMT-related markersFirstly,the composition and stability of the compounds in SQYSF-containing serum were analyzed by high performance liquid chromatography mass spectrometry(HPLC-MS).A mouse podocyte line(MPC-5)was used as the study target.After HG stress,SQYSF-containing serum and Cana intervention were administered,and the survival rate,migration force,apoptosis rate and morphological changes of podocytes in each group were detected by CCK-8 experiment,cell scratch assay,flow cytometry and cell morphology assay,respectively;RT-qPCR and Western blot were performed to detect the effects of signaling axes KDM6 A and KLF10,EMT-related markers α-SMA,N-cadherin,P-cadherin,and the expression of nephrin mRNA and protein,a marker of podocyte damage,in podocytes;RT-qPCR was performed to detect the expression level of miR-378d;RT-qPCR was performed to detect the expression level of miR-378 d.(2)Mechanism of action of SQYSF-containing serum in interfering with EMT in podocytes by inhibiting the KDM6A/KLF10 signaling axisAfter HG stress on podocytes,the KDM6 A signaling pathway inhibitor GSK-J4 and SQYSF containing serum were administered,and the survival rate of podocytes in each group was detected by CCK-8 experiment and the apoptosis rate of podocytes in each group was detected by flow cytometry;RT-qPCR and Western blot were performed to detect the effects of signaling axes KDM6 A and KLF10,EMT-related markers α-SMA,N-cadherin,P-cadherin,and the expression of nephrin mRNA and protein,a marker of podocyte damage,in podocytes.(3)Mechanism of action of SQYSF-containing serum to interfere with EMT in podocytes through miR-378 d inhibition of KDM6A/KLF10 signaling axisAfter detecting whether miR-378 d had binding sites with the target gene DKM6 A using the dual luciferase reporter gene assay,the podocytes were transiently transfected with miR-378d(miR-378 d mimic NC,miR-378 d mimic or miR-378 d inhibitor NC,miR-378 d inhibitor),and HG or SQYSF-containing serum was given to intervene,and the survival rate and apoptosis rate of each group of podocytes were detected by CCK-8 experiment and flow cytometry,respectively;RT-qPCR and Western blot were performed to detect the effects of signaling axes KDM6 A and KLF10,EMT-related markers α-SMA,N-cadherin,P-cadherin,and the expression of nephrin mRNA and protein,a marker of podocyte damage,in podocytes.Results:1.in vivo studyPhysiological indices and kidney morphology of mice: after treatment with SQYSF or Cana,the db/db+SQYSF and db/db+Cana groups significantly reduced blood glucose after the 2th week compared with the db/db group mice(P<0.01),but there was no significant change in weight reduction of mice;the db/db+SQYSF and db/db+Cana groups compared with the db/db group mice had edema of kidney and hypertrophy were significantly improved,and the surface glossiness of kidney was restored to different degrees;Biochemical indices of mice: The m Alb of mice in the db/db group was significantly higher from the 2nd week of membrane creation(P<0.01);after treatment with SQYSF and Cana,the m Alb of the db/db+SQYSF and db/db+Cana groups was significantly lower at 4 weeks after drug intervention(P<0.05),but rebounded at week 6,and m Alb decreased to different degrees after week 8(P<0.01);Scr and BUN were significantly higher in the db/db group(P<0.01),and Scr and BUN were significantly lower in the db/db+SQYSF and db/db+Cana groups compared with the db/db group(P<0.01);Renal pathological staining: HE staining results showed that the overall morphology of glomerul the kidney tissues of mice in the db/db group increased,with obvious thylakoid hyperplasia,and after treatment with SQYSF and Cana,the overall morphology of glomeruli in the kidneys of mice in the SQYSF and Cana groups improved significantly and alleviated glomerular thylakoid hyperplasia to different degrees;PAS staining results showed that glomerular glycogen deposition in mice in the db/db group increased,with basement membrane hyperplasia and thylakoid stromal deposition.The PAS staining results showed that the glomerular glycogen deposition,basement membrane hyperplasia and thylakoid matrix deposition were increased in the db/db group,and glycogen deposition was significantly reduced in the db/db+SQYSF and db/db+Cana groups after treatment with SQYSF and Cana,and glomerular basement membrane hyperplasia and thylakoid matrix deposition were significantly reduced in the db/db group;Masson staining results showed that the glomerular collagen fibers and muscle fibers were significantly deposited in the db/db group;after treatment with SQYSF and Cana treatment,the glomerular collagen fibers and myofibers deposition were significantly reduced in the db/db+SQYSF group and db/db+Cana group mice;The results of RT-qPCR and Western blot assay showed that the kidney tissues of the mice in the db/db group contained KDM6 A,KLF10,α-SMA,N-cadherin mRNA and protein expression levels were significantly increased(P<0.01)and P-cadherin,nephrin mRNA and protein expression levels were decreased(P<0.05,P<0.01)in the kidney tissues of db/db+SQYSF and db/db+Cana groups,and KDM6 A,KLF10,α-SMA,N-cadherin mRNA and protein expression levels were significantly reduced(P<0.01),and P-cadherinm,nephrin mRNA and protein expression levels were increased to different degrees(P<0.01),but there was no significant difference in the elevated P-cadherin mRNA expression levels in the SQYSF group(P>0.05).2.In vitro studies(1)Intervention of SQYSF-containing serum on the damage of podocytes under high glucose(HG)stress and EMT-related markers: The results of HPLC-MS analysis showed that the retention time and signal intensity of the mass spectral peaks of SQYSF-containing serum samples were stable and contained a variety of active ingredients;The results of CCK-8 experiment showed that the viability of podocytes in SQYSF-L,SQYSF-M and Cana groups was significantly higher than that in HG group(P<0.01),while the viability of cells in SQYSF-H group was not significantly higher but lower than that in HG group(P>0.05).The SQYSF-M group showed the most significant increase in cell viability,and the results of scratch assay showed that the width of podocytes scratch in SQYSF-L,SQYSF-M,SQYSF-H and Cana groups was significantly reduced and the migration force of podocytes was enhanced compared with that in HG group(P<0.01),and the migration force of podocytes in SQYSF-M group was most significantly enhanced.The results of flow cytometry showed that the apoptosis rate was significantly lower in the SQYSF-L,SQYSF-M,SQYSF-H and Cana groups compared with the HG group(P<0.01),and the apoptosis rate of podocytes was most significantly reduced in the SQYSF-M group;the results of cell morphology showed that under normal conditions,the podocytes were long shuttle-shaped,with clear cytosol shape and irregular dendritic branching foot protrusions,which protruded from the cytosol.Under normal conditions,the podocytes were long and spindle shaped with well defined cytosol and irregular dendritic branching peduncles that protruded from the cytosol to form intercellular connections.After the intervention of SQYSF and Cana,the cell morphology was significantly improved,and the podocytes morphology was restored to varying degrees,the structure of the peduncle was clearer and the number of peduncles increased,and the intercellular connectivity was enhanced.elevated(P<0.01),P-cadherin and nephrin mRNA and protein expression levels decreased(P<0.01),but P-cadherin mRNA expression level changes were not statistically significant(P>0.05),and KDM6 A,KLF10,α-SMA,N-cadherin mRNA and protein expression was significantly decreased(P<0.01),the expression levels of P-cadherin,nephrin mRNA and protein were increased(P<0.05,P<0.01),and Cana group showed no statistically significant changes in P-cadherin mRNA expression levels(P>0.05).(2)Mechanism of action of SQYSF-containing serum in interfering with EMT in podocytes by inhibiting the KDM6A/KLF10 signaling axis: GSK-J4,an inhibitor of KDM6 A,was used to inhibit KDM6 A.the results of CCK-8 experiment showed that the podocytes viability was significantly lower in HG group(P<0.01)and significantly higher in GSK-J4 and SQYSF groups(P<0.01)compared with HG group,and the difference between the two on promoting podocytes viability(P>0.05).Flow cytometry results showed that the rate of podocytes apoptosis was significantly higher in the HG group(P<0.01)and significantly lower in the SQYSF and GSK-J4groups(P<0.01);RT-qPCR and Western blot assay showed that the expression levels of KDM6 A,KLF10,α-SMA,N-cadherin mRNA and protein expression levels were increased(P<0.01,P<0.05),P-cadherin mRNA and protein expression levels were decreased(P<0.01),nephrin protein expression was decreased(P<0.01),and KDM6 A,KLF10,α-SMA,N-cadherin mRNA expression in SQYSF group and inhibitor GSK-J4 group significantly decreased(P<0.01),among which,the change of KLF10 protein expression was not statistically significant in the GSK-J4 group(P>0.05),P-cadherin mRNA and protein expression levels were elevated high(P<0.01),and nephrin protein expression was significantly increased(P<0.01).(3)Mechanism of action of SQYSF-containing serum to interfere with EMT in podocytes through miR-378 d inhibition of KDM6A/KLF10 signaling axis: The results of dual luciferase reporter gene assay showed that miR-378 d had binding sites with KDM6 A 3’-UTR.RT-qPCR assay showed that the expression of miR-378 d was significantly decreased in the podocytes of HG group(P<0.01)and significantly increased in the podocytes of SQYSF group(P<0.01).For positive and negative validation,miR-378 d mimic and miR-378 d inhibitor were transiently transfected;RT-qPCR assay showed that the expression levels of miR-378 d in miR-378 d mimic NC group and miR-378 d inhibitor NC group were not significantly different compared with N group,and there was no difference(P>0.05),The expression level of miR-378 d was slightly expressed elevated in the miR-378 d mimic group,but no difference(P>0.05),and the expression level of miR-378 d was significantly reduced in the miR-378 d inhibitor group compared to the N group(P<0.01),which was considered as successful miR-378 d transfection;The results of CCK-8 experiment showed that the viability of podocytes in HG+miR-378 d mimic NC group was significantly lower than that in miR-378 d mimic NC(P<0.01),while the viability of podocytes in HG+miR-378 d mimic group was significantly higher(P<0.01),the survival rate of podocytes in miR-378 d inhibitor group was significantly lower than that in miR-378 d inhibitor NC group(P<0.01),while the podocytes viability was significantly higher after SQYSF intervention(P<0.01);Flow cytometry results showed that the podocytes apoptosis rate was significantly higher in the HG+miR-378 d mimic NC group compared with miR-378 d mimic NC(P<0.01),the podocytes apoptosis rate was significantly lower in the HG+miR-378 d mimic group(P<0.01),the podocytes apoptosis rate was significantly higher in the miR-378 d inhibitor group compared with miR-378 d inhibitor NC was significantly higher(P<0.01),and the rate of podocytes apoptosis was significantly lower(P<0.01)after the administration of SQYSF intervention;RT-qPCR showed that KDM6 A,KLF10 and N-cadherin mRNA expression were significantly higher in the HG+miR-378 d mimic NC group compared with miR-378 d mimic NC(P<0.01),but there was no difference in elevated N-cadherin mRNA expression(P>0.05)and P cadherin mRNA expression levels were significantly lower(P<0.01),KDM6 A,KLF10,α-SMA and N-cadherin mRNA expression were significantly lower(P<0.01)and nephrin mRNA expression levels were significantly higher(P<0.01)in the HG+miR-378 d mimic group,Compared with the miR-378 d inhibitor NC group,the expression of KLF10 was significantly higher in the miR-378 d inhibitor group(P<0.01),KDM6 A and N-cadherin mRNA expression levels were increased but not different(P>0.05),and P-cadherin mRNA expression levels were significantly lower(P<0.01),the expression of KDM6 A,KLF10,α-SMA and N-cadherin were significantly decreased in the SQYSF group(P<0.01)and the expression level of P-cadherin was significantly increased(P<0.01);Western blot assay showed that there was no significant difference in the expression of KDM6 A protein in the N group and miR-378 d mimic NC group,while the miR-378 d mimic group significantly inhibited the expression of KDM6 A protein(P<0.01),the N group and miR-378 d inhibitor NC group did not significantly promote the expression of KDM6 A protein,while miR-378 d inhibitor group significantly promoted the expression of KDM6 A protein(P<0.01),and compared with miR-378 d mimic NC group,the expression of KLF10,α-SMA and N-cadherin protein was significantly higher in HG+miR378 d mimic NC group(P<0.01),and P-cadherin and nephrin protein expression level was decreased(P<0.01),KLF10,α-SMA,N-cadherin protein expression was significantly decreased(P<0.01),P-cadherin,nephrin protein expression level was significantly increased(P<0.01)in HG+miR-378 d mimic group,compared with miR-378 d miR-378 d inhibitor NC group,KLF10,α-SMA,N-cadherin protein expression was significantly increased in miR-378 d inhibitor group(P<0.01),P-cadherin,nephrin protein expression was decreased(P<0.01),but no difference in nephrin protein expression was decreased in miR-378 d inhibitor group(P>0.05),KLF10,α-SMA,N-cadherin protein expression was significantly decreased(P<0.01)and P-cadherin,nephrin protein expression level was significantly increased(P<0.01)in SQYSF+miR-378 d inhibitor group.Conclusions: Suoquan Yishen Formula effectively improved the physiological and biochemical indexes and renal pathological changes in DKD mice,alleviated the damage of mouse kidney,and regulated the EMT of mouse glomerular podocyte by intervening miR-378 d to inhibit the expression of KDM6A/KLF10 signaling axis. |
PDF Full Text Request