Experimental Study On Effect And Mechanism Of Trifluoperazine On Mesangial Cell Proliferation

Mesangial cell(MC)plays an essential role in physiological function,for example,regulation of intraglomerular capillary flow,phagocytosis and removal of foreign bodies,secretion of cytokines and generation of Extracellular matrix(ECM).But the abnormal proliferation of MC under the influence of various stimulatory can give rise to kidney damage by releasing inflammatory factors,and ECM components,ultimately leading to glomerular sclerosis and interstitial fibrosis.Furthermore,most patients may develop end-stage renal disease(ESRD)on account of renal fibrosis after many years.Thus inhibition of mesangial cell proliferation has become an important therapy in proliferating glomerular diseases.The present study was planned to investigate the effect and mechanism of trifluoperazine on human mesangial cells in vitro.Meanwhile the effect and mechanism of trifluoperazine on mesangial cells in lupus nephritis model mice were also explored in the study.And the effect of trifluoperazine combined with cyclophosphamide on mesangial proliferative diseases were further studied.Part 1 The influence of trifluoperazine on the abnormal proliferation of mesangial cell in vitro and in vivoObjective: The present study was to confirm the effect of trifluoperazine on the abnormal proliferation of mesangial cells.Methods:(1)The experiment in vitro: Effects of different trifluoperazine concentrations and treatment duration on cell proliferation were analyzed using the MTT assay.(2)The experiment in vivo: HE,PAS and MASSON were used to observe the proliferation of renal mesangial cells in each group.The expression of Ki67 and PCNA were detected by immunohistochemistry and Western Blotting.The serum levels of serum creatinine and blood urea nitrogen were measured by ELISA,in order to test renal function in each group.Results:(1)The experiment in vitro: MTT results showed that high concentrations of serum 20%FBS can stimulate the proliferation of mesangial cells in a dose-and time-dependent manner.After TFP treatment,cell proliferation was significantly lower than that in 20% FBS abnormal group dose-and time-dependently.(2)The experiment in vivo: The results of Hematoxylin and eosin(H&E),PAS,and MASSON staining indicated diffuse proliferation of mesangial cells and matrix in the glomeruli of LN mice,compared to control,which decreased after 3-month treatment with TFP,which was also validated in the study in Western Blotting and immunohistochemistry results of Ki67 and PCNA.The Elisa results showed that the levels of blood urea nitrogen(BUN)and serum creatinine(Cr)were markedly increased in LN group,compared to the control groups.However,they were significantly decreased in TFP-treated LN mice.Conclusion: Trifluoperazine can inhibit the abnormal proliferation of mesangial cells in vitro and in vivo,and can improve the renal function of mesangial proliferative lupus nephritis mice.Part 2 The effect and mechanism research of trifluoperazine on cell cycle in mesangial cell.Objective: The present study was to investigate the effect of trifluoperazine on the cell cycle of mesangial cells and its mechanismMethods:(1)The experiment in vitro: The cell cycle was detected by flow cytometry in each group.According to the results of flow analysis,the changes of the cell cycle-related proteins were investigated by Western Blotting.(2)The experiment in vivo: The effect of TFP on the expression of cycle-related proteins in mesangial cells was further confirmed by Western Blotting in vivo.Results:(1)The experiment in vitro: The results of flow cytometry showed 20%FBS promoted human mesangial cells from G0/G1 phase to S phase,compared with10%FBS normal group.But after 24-h treatment with TFP in 20 % FBS,the number of MCs in G0/G1 was significantly increased and S phases was significantly decreased respectively,compared to 20 % FBS group.The effect was dependent on the dose of TFP.Western Bloting results showed that trifluoperazine inhibited the expression of Cyclin D1,CDK2,CDK4 proteins and promoted P21 protein,but Cyclin E,CDK6 and P27 proteins expression has little effect.(2)The experiment in vivo: Western Blotting results showed that Cyclin D1,CDK2 and CDK4 proteins were higher in LN group than normal mice group.After giving trifluoperazine,expression of Cyclin D1,CDK2,CDK4 proteins were up-regulated and P21 protein was down-regulated,compared with LN.Immunohistochemical results showed that the expression of Cyclin D1 in LN group was significantly higher than that in normal mice.The expression of Cyclin D1 in LN + trifluoperazine was lower than that of the untreated LN group,the difference was statistically significant.Conclusion: Trifluoperazine can block human mesangial cells from G0/G1 phase to S phase,because it could inhibit the expression of Cyclin D1,CDK2,CDK4 proteins and increase the expression of p21 protein.Part 3 The effect and mechanism research of trifluoperazin on the apoptosis of mesangial cellsObjective: The present study was planned to investigate the effect of trifluoperazine on the apoptosis of mesangial cellsMethods:(1)The experiment in vitro: The effect of TFP on the apoptosis of mesangial cells was detected by flow cytometry.The expression of apoptosis-related proteins was detected by Western Blotting and RT-PCR.(2)The experiment in vivo: Western blotting and immunohistochemistry were used to detect the expression of apoptosis-related proteins.Result:(1)The experiment in vitro: The results of flow cytometry showed that the percentage of apoptotic cells in group N(Normal group)was significantly higher than that in Con group(20%).The percentage of apoptotic cells was significantly increased after 24 hours of intervention with TFP,compared with Con.Real-time PCR results showed that the expression of BAX m RNA in group N was higher than that in Con group,and the expression of Bcl-2 m RNA was lower than that in Con group.After 24 hours of intervention with trifloprazine,we observed that the expression of BAX m RNA was significantly higher than that of Con group,while Bcl-2 m RNA significantly lowerthan that of Con group.The changes in protein levels monitored by Western Blotting were consistent with changes in gene levels.while the expression of Bcl-2 m RNA was lower.The expression of Bcl-2 m RNA was significantly higher than that of normal group,the expression of Bcl-2 m RNA was significantly lower than that of the control group,and the expression of Bcl-2 m RNA was significantly higher than that of the control group.The difference was statistically significant.Western Blotting showed that the change of protein level was consistent with the change of gene level.(2)The experiment in vivo: The results of immunohistochemistry showed that the expression of BAX protein in LN group was higher than that in group N,while the expression of Bcl-2 protein also increased,the difference was statistically significant.3 months after the administration of trifluoperazine,we can see that the expression of BAX protein was significantly higher than that of LN group,and the expression of Bcl-2 protein was decreased than LN group.The difference was statistically significant.The ratio of BAX/ Bcl-2 was lower in LN,compared with that in N.The ratio of BAX / Bcl-2 was higher in LN+TFP,compared with that in LN.Conclusion: Trifluoperazine can induce apoptosis of abnormal proliferated mesangial cells both in vitro or vivo,by inhibiting the expression of Bcl-2 and promoting the expression of BAX.Part 4 Trifluoperazine affects cell cycle and apoptosis through P-JNK and p-AKT pathwaysObjective: The present study was further to study the mechanism of trifluoperazine on the proliferation of mesangial cells,and its effect on signal transduction pathways P13 K / Akt,MAPK / JNK and MAPK / ERK1 / 2.Methods: Western blotting was used to detect the expression of pathways-related proteins.Result: Western Blotting results showed that 20% high concentration of fetal bovine serum could increase the expression of p-JNK,p-AKT and p-ERK protein compared with 10% FBS normal cell control group(Normal).After treatment with TFP,the expression of p-JNK,p-AKT was down-regulated compared with the results of the 20% FBS group.At the same time,we can observe that the expression of p-ERK protein was not significantly different from that of 20% FBS.Western Blotting results showed that the expression of p-JNK,p-AKT and p-ERK protein was significantly increased after PDGF stimulation comared normal cell control group.The expression of p-JNK and p-AKT was down-regulated compared with that of PDGF group,but p-ERK protein was no change in PDGF + TFP group.Conclusion: TFP suppressed p-AKT and p-JNK without affecting p-ERK1/2,affecting apoptosis and cell cycle in mesangial cell.Part 5 Effects of trifluoperazine combined with cyclophosphamide on mice with lupus nephritisObjective: The aim of present study was to observe whether the combination of trifluoperazine and cyclophosphamide could better inhibit the proliferation of mesangial cells,and effect on liver and kidney function in mice with lupus nephritis,compared with monotherapy.Method:HE,MASSON staining were used to observe the proliferation of renal mesangial cells in each group.Western Blotting was used to detect the expression of Ki67 and PCNA in each group.The serum levels of serum creatinine,urea nitrogen aspartate minotransferase(AST)were measured by Elisa method.Result: HE and PAS staining showed that the renal tissue of N group was normal;The proliferation of mesangial cell was obvious in the LN group.Mesangial cell proliferation in LN + TFP group and LN+ CTX group was lower than that of the LN group.Compared with LN group,the degree of mesangial cell proliferation was further decreased in LN + 1/2TFP + 1/2CTX group.Western blotting results of Ki67 and PCNA in LN group were significantly higher than those in normal group.The expressions of Ki67 and PCNA were consistent with changes above.The results of Elisa study showed that the level of AST in LN group,LN + TFP group,LN + CTX group and LN + 1/2TFP + 1/2CTX group was all increased significantly compared with N group,which was statistically significant.Compared with LN group,the level of AST was no significant difference whether in LN + TFP group or LN + 1/2TFP + 1/2CTX group,while that was significantly increased in LN + CTX group.TFP on liver function damage is small or no,CTX easily lead to liver damage,1/2TFP + 1/2CTX on liver damage is also smaller.Conclusion: Combination of TFP and CTX,with semi-dose respectively,is better than using single drug on the effect of inhibiting mesangial cell proliferation in lupus nephritis mice.And combination of TFP and CTX,with dosage by half,has less adverse reactions than the single high dose CTX.