| PART 1. Research on Protective Function of Hydrogen Rich Water in UUO ModelAim:This research is designed to study whether hydrogen rich water is able to prevent kidney from UUO insult.Methods: The animal model of renal fibrosis was induced by Unilateral Ureteral Obstruction(UUO) in balb/c mouse. They were divided into four groups: Normal group, Sham group, UUO group and UUO + hydrogen rich water(HW) group. Their right kidneys and blood were harvested on the 10 th day after operation. Serum creatinine(Scr) and blood urea nitrogen(BUN) were tested, H.E. trichome were performed to examine the morphological changes in kidney. Meanwhile, immunohistochemical staining of tissue section line was used to exhibited the distribution of α-SMA of each group. Additionally, Western-blot was performed to examine the expression of α-SMA of each group.Results: On the animal model, glomerular and renal interstitial of the Normal group and Sham group changed little. Nevertheless, in UUO group, it was observed that the fibrosis of interstitial along with shrink of glomerular, hyperplasia of basement membrane was significant and part of basement membrane showed their fracture. However, when these changes happened to HW+ UUO group, they became moderate. Immunohistochemical staining exhibited the abundant α-SMA in renal interstitial and glomerular basement membrane from UUO group. And in HW + UUO group, little area of renal interstitial was stained along with parts of glomerular basement. The examination of SC and BUN showed the obviously increase of BUN(BUN P<0.05) along with a certain increase of Scr(Scr P<0.1) in UUO group when compared with Normal group. And the difference between the increase of UUO group and HW+ UUO also make sense(Scr P<0.05; BUN P<0.05). Compared with Normal group, α-SMA was significantly increased in UUO group( P<0.05). In UUO+HW group, α-SMA showed its mild increase in comparisom with Normal group(P > 0.05), and showed its distinct decrease when compared with UUO group(P<0.05).Conclusion: HW is able to prevent kidney from UUO insult.PART 2. Experiment of the Mechanism on Hydrogen Rich Water Preventing HK-2 Cells From TGF-β1-induced Renal FibrosisAim:This research is designed to study the hydrogen rich water’s ability of anti-肾纤维化 via Sirt-1, and investigate whether hydrogen rich participate in the influence on typical TGF-β1/ Smad2 pathway.Method: HK-2 cells were divided into four groups: Control group, HW group, TGF-β1 group, HW+ TGF-β1 group. Western-blot was performed to examine the expression of α-SMA, Fibronectin, E-cadherin, Smad2 of HK-2 cells from each group. Additionally, Sirt-1 was also analyzed. Furthermore, Sirtinol was used to inhibit the expression of Sirt-1 of HK-2 from HW+ TGF-β1 group, following with the detection of α-SMA, Fibronectin, E-cadherin to prove whether HW impact on renal fibrosis via the influence of Sirt-1.Results:Fibrosis markers α-SMA and fibronectin were significant up-regulated(α-SMA P<0.05;Fibronectin,P<0.05) and E-cadherin were down regulated obviously(P<0.05) in TGF-β1 group compared with HW+TGF-β1 group. Then TGF-β1/Smad2 pathway was tested. In HW+ TGF-β1 group, Smad2 expressed the similar level in comparison with that in TGF-β1 group(P>0.05). In comparison with Normal group, Sirt-1 exhibited its similar expression(P>0.05) in HW group, while in TGF-β1 group, it presented its declining(P<0.05). Compared with TGF-β1 group,significant rising expression of Sirt-1 was appeared in HW+ TGF-β1 group(P<0.05), but it was still a little lower than that in Normal group(P>0.05). After using sirtinol, E-cadherin expressed similarly in HW+ TGF-β1 group compared with TGF-β1 group(P>0.05).Conclusion: HW is able to protect kidney from fibrosis insulted via Sirt-1, but we have no evidence to document that the guidance is intervening the typically TGF-β/Smad2 pathway. |
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