Inhibition Of Zhenqing Recipe On SREBP-1 Expression In Rat Mesangial Cells Cultured With A High Glucose Media

Objective:We assumed that Zhen Qing Recipe(ZQR) would inhibit proliferation and accumulation of intracellular triglyceride content through suppression of sterol regulatory element-binding protein-1(SREBP-1) expression in rat mesangial cells cultured with a high glucose media and would exert a favorable effect against diabetic nephropathy(DN).Methods:The rat mesangial cells(HBZY-1) were cultured in DMEM and routinely growth-arrested without FBS for 24 h in 5.5 mM D-glucose. In some experiments, the cells were exposed to 25mM glucose (HG group) or with 4mM metformin (MF group) or with 50μg/ml ZQR (ZQR group) for 48h. Also the mesangial cells were cultured in 5.5mM glucose concentration (normal group) for 48h. After that:1.Cell proliferation was monitored by MTT and flow cytometry (FCM).2. Intracellular triglyceride(TG) content was examined by ELISA.3. TGF-β1 and FN secretion and protein expression were measured by ELISA and immunohistochemistry, respectively.4. SREBP-1, ACC and FAS mRNA levels were detected by RT-PCR.5. The protein expression of SREBP-1, ACC and FAS were detected by western blot.Results1. The cell proliferation by MTT and flow cytometry: the proliferation of mesangial cells were significantly increased in 25mM glucose medium for 48h compared with the normal group (P <0.05). Both the ZQR group and the MF group markedly inhibited cells proliferation compared with the HG group (P <0.05) and the two groups were almost identical to that seen in cells proliferation (P >0.05).2. TG content comparison of cells in each group: After 48h, cells grown in a medium with 25mM glucose had high levels of lipid amount as compared to normal group(P <0.01). A striking reduction in intracellular triglyceride accumulation was observed in mesangial cells treated with ZQR(P <0.01) and also MF was effective in preventing fat accumulation(P<0.05) as compared to HG group. This data collectively showed that ZQR could reverse intracellular TG accumulation with 50μg/ml exerting a desirable effect.3. TGF-β1 and FN secretion and protein expression by ELISA and immunohistochemistry. (1) TGF-β1 changes of each group: there was a marked increase of total TGF-β1 in 25mM glucose medium as compared with mesangial cells incubated in 5.5mM glucose(P <0.01). However, the magnitude of increase in 25mM glucose had been blunted by ZQR or MF. After 48h of culture with ZQF, mesangial cells displayed a significant decrease of TGF-β1 compared with HG group (P <0.05). Cells cultured with MF also demonstrated a marked reduction in TGF-β1 secretion (P <0.01). (2) FN levels of each group: rat mesangial cells after incubation with 25mM glucosefor 48h exhibited enhanced FN protein expression and FN secretion in cell culture supernatant compared with normal group (P <0.01). However, in the presence of ZQR, the response was abrogated. ZQR inhibited FN levels (P <0.01), and also MF diminished extracellular matrix protein FN production and secretion compared with HG group (P <0.01).4. SREBP-1, ACC and FAS mRNA levels by RT-PCR in each cell group: 25mM glucose increased SREBP-1, ACC and FAS mRNA levels compared with normal group(P <0.01). But both ZQR and MF attenuated the increased expression of SREBP-1, ACC and FAS induced by 25mM glucose (P <0.01 or P <0.05). No difference in lipogenic genes mRNA expression was observed with MF or ZQR treatment.5. SREBP-1, ACC and FAS protein expression by Western blotting: 25mM glucose increased SREBP-1, ACC and FAS protein expression compared with normal group(P <0.01). However, when rat mesangial cells were treated with ZQR (50μg/ml) or MF (4mmol/L) for 48h in 25mM glucose, SREBP-1, ACC and FAS expression were no longer elevated compared with HG group (P <0.01).Conclusion:Our investigation findings demonstrated that ZQR diminished SREBP-1 with attenuation of intracellular TG content and cell proliferation, also associated with lowered TGF-β1 expression. This suggests that SREBP-1 could be a possible mechanism by which ZQR prevents mesangial expansion and glomerulosclerosis and improves diabetic renal function.