| Objective: Diabetic nephropathy(DN) is one of the most common and serious microvascular complications of diabetes mellitus(DM) and the leading cause of end stage renal disease. Inflammatory response of renal inherent cells played an important role in the occurrence and development of diabetic kidney damage. TGF-β1/p38 MAPK pathway is one of the most important inflammatory response pathways. TGF-β1 played a role of inflammatory response by activating gene sequences of p38 MAPK and promoting the expression of downstream nuclear transcription factor-kappa B and monocyte chemotactic protein 1. Blocking or inhibiting inflammation, relieving renal fibrosis become the key to the prevention and treatment of diabetic nephropathy. Hydrogen is electrically neutral and much smaller than the oxygen molecules, it can easily penetrate cellular and intracellular membranes. Hydrogen has been shown to suppress inflammatory response and apoptosis in organ injuries. Both the inhalation of hydrogen gas and the application of hydrogen rich saline(HRS) are effective routes for utilisation of the therapeutic properties of hydrogen, as certified by prior experimental and clinical studies. Studies have reported that hydrogen rich saline can also slow down the progress of in diabetic nephropathy, but the exact mechanism remains unclear.In this study, we established the streptozotocin(STZ)-induced diabetic rats model and estimate the inflammatory reaction by measuring the expression of TGF-β1, p-p38 MAPK, NF-κB, MCP-1. Further we observed the possible intervention mechanism of hydrogen rich saline(15ml/Kg) on it to provide experimental evidence for the prevention and treatment of renal injury of diabetes.Methods:36 healthy male SD rats of clean level, with weight of 180 ~ 220 g were selected and randomly divided into normal control group(NC group, n =12), diabetic model group(DM group, n=12), hydrogen rich saline treatment group(DM+HRS group, n=12). After 1 week adaptive feeding, the DM and DM+HRS group rats should be fast intraperitoneal injected of STZ 65 mg/kg(dissolved in o.1 mol/L sodium citrate buffer, PH4.3) to be diabetic rats. After 72 h of intraperitoneal injection, sample caudal vein blood, measure the blood glucose with roche glucose meter. Only rats with blood glucose concentration≥16.7mmol/L, urine sugar +++~++++ were used. NC group the amount of citric acid buffer with an empty belly. All animals were housed with water and food available adlibitum with temperature(20 ℃~ 26 ℃) and humidity(40%~70%) relatively constant. DM+HRS group rats were lavaged with 15 ml/kg hydrogen rich saline, and NC group, DM group the same amount of saline every day. At 8th and 12 th weeks after the onset of diabetes, 6 rats were sacrificed randomly for each group respectively and measure their 24 h urine albumin, blood glucose, serum creatinine(Scr) and blood urea nitrogen(BUN), the renal cortical tissues were removed and used for detecting rat kidney pathological changes under the light microscopy, the expression of TGF-β1, p-p38 MAPK, NF-κB and MCP-1 with immunohistochemistry and realtime fluorescent quantitative PCR(RTFQ-PCR). All the experimental data were expressed as mean ± standard deviation((?)±S), one-way analysis of variance was used in date analysis using SPSS 13.0. It was considered statistically significant when P-value was less than 0.05.Results:1 General situationFood-intake and urine of NC group had no significant change, the fur was lustrous, the reaction was agile and the weight increased obviously. Compared with the same period in the NC group, the DM+HRS group and DM group appeared polydipsia, polyphagia and urorrhagia gradually. The fur became dry and the weight decreaaed obviously, the difference was statistically significant (P < 0.05).The weight of DM and DM+HRS group had no statistical significance at two time points(P>0.05).2 Biochemical indicators2.1 Blood glucoseCompared with the same period in the NC group, the blood glucose of both DM and DM+HRS group of rats was increased, the difference was statistically significant(P<0.05), Compared with the same period in the DM group, the blood glucose of rats was reduced in DM+HRS group, the difference was statistically significant(P<0.05).2.2 Serum creatinine and blood urea nitrogenCompared with the same period in the NC group, the serum creatinine, blood urea nitrogen of DM and DM+HRS group rats were significantly increased, the difference was statistically significant(P<0.05). Compared with the same period in the DM group, the rat serum creatinine and urea levels were reduced in DM+HRS group, the difference was statistically significant(P<0.05).2.3 24 hours urinary proteinCompared with the same period in the NC group, the 24 hours urinary protein of DM and DM+HRS group of rats was increased remarkable, the difference was statistically significant(P<0.05), this change was more pronounced at DM group(P<0.05).3 Histochemical changesFrom Light microscope in kidney tissue of each group, HE and PAS staining revealed that the tissue of rats in NC group showed no obvious pathological changes throughout the experiment, Compared with the same period of NC group, DM and DM+HRS group of glomerular volume increased, glomerular mesangial matrix was increased, mesangial cells increased, basement membrane thickened. The pathological change was more obvious at 12 weekends. Compared with the same period in the DM group, kidney pathological structure of DM+HRS group was improved at the same time.4 The immunohistochemicalCompared with the same period in the NC group, TGF-β1, p-p38 MAPK, NF-κB and MCP-1 expression in rats kidney tissues of DM and DM+HRS group increased, the difference was statistically significant(P<0.05). While expression level of DM+HRS group was lower than the same period of DM group, the difference was statistically significant(P<0.05).5 Realtime fluorescent quantitative PCRCompared with the same period in the NC group, TGF-β1 m RNA and MCP-1 m RNA expression in rats kidney tissues of DM and DM+HRS group increased, the difference was statistically significant(P<0.05). Compared with the same period in the DM group, TGF-β1 m RNA and MCP-1 m RNA expression in DM+HRS group declined, the difference was statistically significant(P<0.05).Conclusions:1 TGF-β1, p-p38 MAPK, NF-κB and MCP-1 expression of diabetic kidney are significantly increased, which show that inflammation is obvious in diabetic rats kidney tissue.2 Hydrogen rich saline can reduce the expression of TGF-β1, p-p38 MAPK, NF-κB and MCP-1, thus alleviating the inflammation and fibrosis, protecting the renal function. Its mechanism may be related to inhibition of the TGF-β1/p38 MAPK pathway. |
PDF Full Text Request