| PartⅠObjective:Previous literatures have reported that Cryptotanshinone (CTS) have the abitity to alleviate cardiac fibrosis, but how about its effect on kidney fibrosis? This part mainly investigates whether CTS could ameliorate kidney injury and tubulointerstitial fibrosis.Methods:40 mice were employed in the study and randomly ranged into four groups (n=10):Sham operation+Vehicle; Sham operation+50mg/kg/d CTS; Unilateral ureteral occlusion (UUO)+vehicle; UUO+50mg/kg/d CTS. All the mice were sacrificed 7 days after UUO. Tubular injury were assessed by HE staining; collagens deposited in the interstitial were measured by Masson’s staining and extracellular matrix were assessed using Immunohistochemistry stainging. Cytotoxic and proliferative effect of CTS on rat renal tubular epithelial cell (NRK52E) were evaluated by CCK-8 method. NRK52E cells were cultured in virto and treated with control,5ng/ml TGF-β1,5ng/ml TGF-β1+10uM CTS or 5ng/ml TGF-β1+20uM CTS. Western blot and Real-time PCR were performed to detected the protein and mRNA levels of Collagen-1 and Fibronectin.Results:The results of CCK-8 method indicated there was no difference between control and CTS treated group for cell viability. H&E staining revealed tubular injury score in UUO plus vehicle treated mice group was 2.30±0.35, and the proportion of deposited interstitial collagens evaluated by masson staining was 13.95±1.68; while tubular injury score in UUO plus 50mg/kg/d CTS treated mice group was 0.96±0.25, and proportion of deposited interstitial collagens was 7.38±1.28; These data had statistical difference between these two groups, P<0.05. The results of western bolt and IHC indicated the expressions of Collagen-1 and Fibronectin were significantly increased after 7 days of UUO, while treatment of CTS inhibited the expressions of Collagen-1 and Fibronectin after UUO; there was statistical difference between these two groups, P<0.05. The results of western blot and Real-time PCR indicated fibronectin and collagen-1 production over-expressed upon the TGF-β1 treatment, but these effects were alleviated by the 10uM and 20uM CTS; there are statistical difference between these groups.Conclusions:CTS could attenuate tubular injury and interstitial fibrosis induced by UUO, so as to improved kidney fibrosis.Part IIObjective:Epithelial-mesenchymal transition (EMT) is one of the most important machanisms in kidney fibrogenesis, so this part aims to investigate whether CTS could hinder Epithelial-mesenchymal transition.Methods:UUO mice model was established in vivo, and the mice were treaed with vehicle or 50mg/kg/d CTS. Immunohistochemistry stainging and Western blot were performed to detect expressions of a-SMA and E-cadherin in different groups. NRK52E cells were cultured in virto and treated with control,5ng/ml TGF-β1,5ng/ml TGF-β1+10uM CTS or 5ng/ml TGF-β1+20uM CTS, then expressions of a-SMA and E-cadherin were observed using immunofluorescence staining 24h after the treatment, and the protein levels of a-SMA and E-cadherin were measured by western blot.Results:The results of western bolt and IHC indicated the expressions of a-SMA and E-cadherin were significantly increased after 7 days UUO, while treatment of CTS could reverse these effects; there were statistical difference between these two groups, P<0.05. The results of immunofluorescence staining and western blot found that α-SMA and E-cadherin production over-expressed upon TGF-β1 treatment, but these effects were alleviated by the 20uM CTS treatment.Conclusions:CTS could inhibited the process of Epithelial-mesenchymal transition.PartⅢObjective:TGF-β1/Smads and MAPK are two important signaling pathways in regulating kidney fibrosis and Epithelial-mesenchymal transition, so this part aims to investigate the underlying mechanism of CTS blocking Epithelial-mesenchymal transition.Methods:Western blot was performed to assessed the relative protein expressions involved in TGFβ/Smads and MAPK signaling pathways. NRK52E cells were cultured in virto and treated with control,5ng/ml TGF-β1,5ng/ml TGF-β1+10uM CTS or 5ng/ml TGF-β1+20uM CTS, then relative protein expressions involved in TGFβ/Smads and MAPK signaling pathways were evaluated by western blot. The necular translocation of Smad2, Smad3 and Smad4 were observed by immunofluorescence staining. Meanwhile, pcDNA3.1(+)-Smad3 and pGL3-integrin β1 promoter plasmids were constructed and then co-transfected into NRK52E cells. Dual luciferase reporter analysis targeting to further verified Smad3 regulating integrin β1 promoter activity. ChIP analysis was used to detectd the direct intractions between Smad3 and integrin β1 promoter sequences.Results:The results of western bolt indicated TGF-β1/Smads and MAPK signals were activated after 7days of UUO, evidenced by up-regulated of p-P38, p-ERK, p-JNK, p-Smad2, p-smad3 and Smad4. Treatment with CTS only inhibited the Smad3 phosphorylation but not Smad2, p38, ERK and JNK, neither the inhibition of Smad4. The in vitro data showed the similar results that CTS could only blocked the activation of Smad3 but had no effects on Smad2 or Smad4. The results of immunofluorescence staining showed 20uM CTS could inhibited the necular translocation of Smad2, Smad3 and Smad4. In addition, Dual luciferase reporter analysis indicated CTS could blocked of activity of integrin β1 promoter activity induced by Smad3. Chip analysis further conformed CTS inhibited Smad3 binding to integrin β1 promoter sequences.Conclusions:1. CTS could depress EMT via inhibiting the phosphorylation of Smad3, in addition to hinder the necular translocation of Smads complex.2. CTS could inhibited transcription of integrin β1 induced by Smad3, and inhibited Smad3 binding to integrin β1 promoter sequences. |
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