Experimental Study On Treatment Of Mesangial Proliferative Glomerular Disease By Targeting Mesangial Cell Cycling With The Combination Of Tacrolimus And Mycophenolate Mofetil

Part 1 The influence of tacrolimus and mycophenolate mofetil on the cell cycle of human mesangial in vitro Objective :The aim of the present study was to investigate the influence of tacrolimus(TAC) and mycophenolate mofetil(MMF) on the cell cycle of human mesangial. Methods:Human mesangial cells(HMC), which were divided into drug group and control group, were cultured for 24 h, 48 h, and 72 h separately. The drug group was cultured HMCs were treated with 1μmol/L 、5μmol/L TAC or 2.5μmol/L、10μmol/L MMF. The activation of the HMC amplification was determined by MTT chromatometry, and the cell cycle and apoptosis of HMC was detected by flow cytometry. Results:After treating with 5 μmol/L TAC for 48 h, the percentage of cells in the S phase decreased by 41% while the percentage of cells in the G0/G1 phase increased. TAC(at 1 and 5 μmol/L) did not significantly induce apoptosis in HMCs over 48 h of treatment. MMF(at 2.5 and 10 μmol/L) inhibited the proliferation of HMCs after 24, 48 or 72 h of treatmentand strongly suppressed the cells entry into the G2/M phase. There was a significant increase in the apoptosis rate of HMCs that were treated for 48 h with MMF(at 2.5 and 10 μmol/L) and the percentage of apoptotic cells increased in a dose-dependent manner. The apoptosis rate of HMCs that was higher after treating for 48 h with 10μmol/L MMF.Conclusions:TAC can block the progression of HMCs from G1 phase into S phase, and MMF can block the progression of HMCs from S phase into G2 phase. The two drugs can block the progression of HMCs in different cell cycle phases. Part 2 The mechanism of TAC and MMF inhibiting periodic proliferation of HMCsObjective :The aim of the present study was to investigate the mechanism of TAC and MMF inhibiting periodic proliferation of HMCs. Methods:The experiment 1, human mesangial cells(HMCs) were divided into control(CTL) group, TGF-β1 group, CTL+TAC group and TGF--β1+TAC group. The experiment 2, human mesangial cells(HMCs) were divided into control group, PDGF group, CTL+MMF group and PDGF+MMF group. Western-blotting was used to detect the expression of Smad2 and the level of p38 phosphorylation in different groups. Results:The results of Western-blotting showed in the part 1: compared with CTL group, the level of Smad 2 expression significantly raised in TGF--β1 group(p<0.01); compared with CTL group, the level of Smad 2 expression significantly decreased in CTL+TAC group(p<0.01); compared with TGF--β1 group, the level of Smad 2 expression significantly decreased in TGF--β1+TAC group(p<0.01). The results of Western-blotting showed in the part 2: compared with CTL group, the level of PP38/P38 expression significantly up-regulated in PDGF group(p<0.01); compared with CTL group, the level of PP38/P38 expression significantly down-regulated in CTL+MMF group(p<0.01); compared with PDGF group, the level of PP38/P38 expression significantly down-regulated in PDGF+MMF group(p<0.01).Conclusions:TAC inhibits HMCs proliferation by influencing the TGF--β1/Smad2 signaling pathways. And MMF inhibits HMCs proliferation by influencing the p38 signaling pathways. Part 3 According to the cell cycle combination of tacrolimus and mycophenolate mofetil inhibit human mesangial cell proliferation in vitro Object:The aim of the present study was to investigate combination of tacrolimus(TAC) and mycophenolate mofetil(MMF) inhibit human mesangial cell proliferation whether is better than using single drug in vitro. Methods:Human mesangial cells(HMCs) were divided into control(CTL) group, PDGF group, PDGF+T group, PDGF+M group and PDGF+T+M group. The part 2, human mesangial cells(HMCs) were divided into control group, PDGF group, CTL+MMF group and PDGF+MMF group. Western-blotting was used to detect the expression of Smad2 and the level of p38 phosphorylation in different groups.CTL group was cultured by the common culture for 48h; PDGF group was cultured by the common culture contain 20ng/ml PDGF for 48h; PDGF+T group was cultured by the common culture contain 20ng/ml PDGF for 24 h, then was cultured by the common culture contain 5μmol/L TAC for 24 h. PDGF+M group was was cultured by the common culture contain 20ng/ml PDGF for 24 h, then was cultured by the common culture contain 10μmol/L MMF for 24h; PDGF+T+M group was was cultured by the common culture contain 20ng/ml PDGF for 24 h, then was cultured by the common culture contain 2.5μmol/L TAC and 5μmol/L MMF for 24 h. Immunofluorescence staining was used to detect the Ki67 positive proportion of the nucleus and western-blotting was used to detect the expression of Cyclin D1 in different groups. Results:Immunofluorescence staining showed that: compared with CTL group, the positive proportion of Ki67 significantly raised in PDGF group(p<0.01); compared with PDGF group, the positive proportion of Ki67 significantly decreased in PDGF+TAC group and PDGF+MMF group separately(p<0.01); compared with PDGF+TAC group and PDGF+MMF group, the positive proportion of Ki67 significantly decreased in PDGF+TAC +MMF group; and there was no significantly difference in PDGF+TAC group and PDGF+MMF group.The results of Western-blotting showed: compared with CTL group, the level of Cyclin D1 expression significantly up-regulated in PDGF group(p<0.01); compared with PDGF group, the level of Cyclin D1 expression significantly down-regulated in PDGF+TAC group and PDGF+MMF group separately(p<0.01); compared with PDGF+TAC group and PDGF+MMF group, the level of Cyclin D1 expression significantly down-regulated in PDGF+TAC +MMF group(p<0.01). Conclusions:Combination of TAC and MMF and dosage by half is better than using single drug on the effect of inhibiting human mesangial cell proliferation in vitro. Part 4 According to the cell cycle combination of tacrolimus and mycophenolate mofetil inhibit mesangial cells in mice with lupus nephritis proliferation in vitro Object:The aim of the present study was to investigate combination of tacrolimus(TAC) and mycophenolate mofetil(MMF) inhibit mesangial cells in mice with lupus nephritis proliferation whether is better than using single drug in vitro. Methods:Rats were randomized into 5 groups: Wt group, Lupus group, Lupus+TAC group, Lupus+MMF and Lupus+TAC+MMF group. Lupus+TAC group was Lupus mice by intraperitoneal injection of 1mg/kg﹒d TAC; Lupus+MMF group was Lupus mice to use 60 mg/kg﹒d MMF to lavage; Lupus+TAC+MMF group was Lupus mice by intraperitoneal injection of 0.5 mg/kg﹒d TAC and using 30 mg/kg﹒d MMF to lavage. The rats from Lupus+TAC group, Lupus+MMF and Lupus+TAC+MMF group used different drugs in 3 months. To 6 months of age, 5 groups of experimental animals put to death and then return the kidney tissues. Using PAS staining and MASSON staining, we observed the proliferation of the kidney tissues mesangial cell from 5 groups of experimental animals. Immunofluorescence staining was used to detect the the expression of Ki67 and PDGF, and western-blotting was used to detect the expression of Cyclin D1 and PDGF in different groups. Results:PAS staining and MASSON staining showed that: the mesangial cells of Lupus mice hyperplasia were obvious. Compared with Lupus group, the mesangial cells of Lupus mice hyperplasia get better in Lupus+TAC group and Lupus+MMF group; compared with Lupus+TAC group and Lupus+MMF group, the mesangial cells of Lupus mice hyperplasia get better in Lupus+TAC+MMF group separately.Immunohistochemical showed that: compared with Wt group, the expression of Ki67 and PDGF significantly increased in Lupus group(p<0.01); compared with Lupus group, the expression of Ki67 and PDGF significantly decreased in Lupus+TAC group and Lupus+MMF group separately(p<0.01); Conclusions:Combination of TAC and MMF and dosage by half is better than using single drug on the effect of inhibiting mice with lupus nephritis mesangial cell proliferation in vitro. Part 5 The combination of tacrolimus and mycophenolate mofetil inhibits the proliferation of mesangial cells of lupus nephritis by regulating cell cycle Objective :The aim of the present study was to investigate the influence of TAC and MMF inhibiting the proliferation of mesangial cells of nephritis by regulating cell cycle. Methods:Six cases type IV patients with lupus nephritis by kidney biopsy received treatment of the combination of TAC(2mg, twice daily), MMF(500 mg, twice daily) and methyl prednisone for 6 months and follow-up. At the end of the 6-month follow-up period, the six patients received renal biopsy. We compared the change on SLADAI score, 24-hour proteinuria, the levels of serum creatinine and albumin, anti ds DNA antibodies, complement C3 and other clinical indicators before and after thetreatment. And we detected the expression of Cyclin D1, Ki67 and PDGF in renal tissues by using immunohistochemical method. Results:After the combination therapy for 6 months, the SLADAI score, 24-hour proteinuria, the levels of serum creatinine and albumin were significantly improved and the kidney pathological damage reduce. The expression of Cyclin D1, Ki67 and PDGF decreased significantly in the mesangial cells. And there were no serious adverse effects. Conclusions:The combination TAC and MMF can effectively inhibit the proliferation of mesangial cells of lupus nephritis by regulating cell cycle. It is safe in clinical usage with less adverse reaction.