糖肾方含药血清对高糖诱导肾小管上皮细胞增殖作用及对TGF-β1、HO-1干预作用的研究

Study on effect of Tangshen prescription-containing serum on proliferation of renal tubular epithelial cells induced by high glucose and intervention of TGF-β1 and HO-1

关键词:  糖肾方  肾小管上皮细胞  血红素加氧酶-1  转化生长因子β1

Key words:  Tangshen prescription  renal tubular epithelial cell  HO-1  TGF-β1

摘要:

[目的]探讨糖肾方含药血清对高糖诱导肾小管上皮细胞增殖作用及对转化生长因子（TGF）-β1、α-平滑肌肌动蛋白（α-SMA）、血红素氯合酶-1（HO-1）干预作用。[方法]制备小鼠含药血清；培养肾小管上皮细胞分为：空白对照组、高糖组（30 mmol/L葡萄糖）、空白血清组（30 mmol/L葡萄糖+10%空白血清）、糖肾方低剂量血清组（30 mmol/L葡萄糖+10%低剂量含药血清）、糖肾方高剂量血清组（30 mmol/L葡萄糖+10%高剂量含药血清）。使用4-甲基偶氮唑蓝（MTT）法检测糖肾方含药血清对高糖诱导肾小管上皮细胞增殖的影响。运用逆转录聚合酶链式反应（RT-PCR）和蛋白免疫印迹法（Western blotting）检测肾小管上皮细胞中TGF-β1、HO-1和α-SMA蛋白的表达。[结果]高糖诱导下，肾小管上皮细胞呈成纤维细胞样的长梭形，细胞核呈梭形改变。MTT检测：与空白组比较，高糖组光度值明显增高（P<0.05）；与高糖组比较，高低剂量含药血清组在培养24 h后吸光度值均有下降，吸光度差异有统计学意义（P<0.05），且高剂量组光度值下降强于低剂量组（P<0.05）。Western blotting及RT-PCR测定实验结果显示：与空白对照组细胞对比，高糖组中TGF-β1、α-SMA含量增加；经过含药血清干预后，高糖诱导的HK-2细胞TGF-β1、α-SMA含量降低，差别有统计学意义（P<0.05）；糖肾方高、低剂量组血清比较，在降低TGF-β1方面，高剂量组效果较好（P<0.05），在降低α-SMA方面没有统计学差异（P>0.05）。HO-1在高糖诱导后含量稍升高，与空白对照组对比，差异有统计学意义（P<0.05）；在经过糖肾方含药血清干预后，含量升高，与高糖组对比，差异有统计学意义（P<0.05），糖肾方高低剂量组血清比较，升高HO-1含量方面没有统计学差异（P>0.05）。[结论]糖肾方通过抑制高糖诱导的肾小管上皮细胞增殖和干预TGF-β1、α-SMA、HO-1相关分子的表达，可以抑制细胞外基质成分沉积，减少氧化应激反应，减轻细胞损伤，逆转上皮-间质转分化，从而抑制肾间质纤维化，延缓糖尿病肾病病情进展。

Abstract:

[Objective] To investigate the effect of Tangshen prescription-containing serum on the proliferation of renal tubular epithelial cells induced by high glucose and the intervention of TGF-β1, α-SMA and HO-1.[Methods] Rat serum containing drugs was prepared; cultured renal tubular epithelial cells were divided into blank control group, high glucose group (30 mmol/L glucose), blank serum group (30 mmol/L glucose + 10% blank serum), and sugar kidneys Low-dose group (30 mmol/L glucose + 10% low dose drug-containing serum), high-dose sugar kidney group (30 mmol/L glucose + 10% high-dose drug serum). MTT assay was used to detect the effect of Tangshen prescription-containing serum on proliferation of renal tubular epithelial cells induced by high glucose. The expression of TGF-β1, HO-1 and α-SMA protein in renal tubular epithelial cells was detected by RT-PCR and Western blotting.[Results] Induced by high glucose, renal tubular epithelial cells lost their intrinsic morphology, exhibited a fibroblast-like long spindle shape, and showed a fusiform change in the nucleus. MTT assay:compared with the blank group, the photometric value of the high glucose group was significantly higher (P<0.05); compared with the high glucose group, the absorbance values of the high and low dose drug-containing serum groups decreased after 24 hours of culture, and the difference in absorbance was statistically significant(P<0.05), and the photometric value of high-dose group decreased more than that of low-dose group (P<0.05). The results of Western blotting and RT-PCR showed that:compared with the control group, the levels of TGF-β1 and α-SMA in the high glucose group increased; after intervention with the serum containing the drug, the high glucose induced HK-2 cells TGF-β1 The content of α-SMA decreased, and the difference was statistically significant (P<0.05). Compared with the high-dose and low-dose group of Tangshenfang, the effect of high-dose group was better (P<0.05) in reducing TGF-β1, and α-SMA was decreased. There was no difference in the aspects (P>0.05). The contents of HO-1 was slightly increased after induction of high glucose, compared with the blank control group, the difference was statistically significant (P<0.05); after intervention of Tangshen prescription containing serum, the content was increased, compared with the high glucose group The difference was statistically significant (P<0.05). There was no difference in elevated HO-1 levels between the high-dose group and the low-dose group (P>0.05).[Conclusion] Tangshen prescription can inhibit the deposition of extracellular matrix components, reduce the oxidative stress response, and reduce the cells by inhibiting the proliferation of tubular epithelial cells induced by high glucose and interfering with the expression of TGF-β1, α-SMA, and HO-1 related molecules. In terms of injury, the epithelial-mesenchymal transition was reversed, thereby inhibiting renal interstitial fibrosis and delaying the progression of diabetic nephropathy.