ABSTRACT
Aim To investigate the protective effect and mechanism of JTE-013 on allergic rhinitis (AR) by regulating mitochondrial injury and apoptosis through RhoA/ROCKl/Drpl pathway. Methods AR model was established by ovalbumin (OVA) in mice. Nasal tissue sections were then stained with HE, TUNEL and DHE. Western blot assay. In vitro, human nasal epithelial cells (HNEpCs) were stimulated with human recombinant interleukin-13 (IL-13), and the effects of JTE-013 and Y27632-related protein expression were detected by Western blot. Immunofluorescence was used to observe the effects of JTE-013 and Y 27632 on total ROS, mitochondrial membrane potential and mitochondrial ROS generation, Drpl translocation and Cyt-c expression in cells. Results JTE-013 reduced the frequency of nose rubbing and sneezing, reduced nasal mucosal thickening and decreased eosinophil infiltration in AR mice. TUNEL and DHE staining results suggested that JTE-013 could inhibit apoptosis and reduce ROS expression in mouse nasal epithelial cells. Western blot showed that both JTE-013 and Y 27632 could significantly reduce RhoA, ROCK1, Drpl and p-Drpl(616), inhibit the expression of apoptotic proteins Bax, cleaved-caspase-3, Cyt-c, cleavedcaspase-9 and up-regulate the expression of p-Drpl (637) and Bcl-2. Immunofluorescence showed that inhibitors of JTE-013 or ROCK1 almost blocked IL-13mediated increase in ROS and mtROS production, inhibited decrease in mitochondrial membrane potential, and blocked Cyt-c expression and Drpl translocation in nasal mucosal epithelial cells. Conclusion JTE-013 can regulate the morphology and function of mitochondria by inhibiting RhoA/ROCKl/Drpl signaling axis, thereby alleviating nasal epithelial cell inflammation in mice with allergic rhinitis.
ABSTRACT
Objective To investigate the effect and mechanism of salvianolic acid B(SalB)on myocardial fibrosis in diabetic rats based on RhoA/ROCK1 signaling pathway.Methods Diabetic rat model was established by injecting streptozotocin into tail vein of healthy male SD rats.At the end of the 12th week, the collagen fiber expression in the left ventricular myocardium of rats was detected by Sirius Red reagent staining.The protein expressions of α-SMA, Collagen and Collagen III in left ventricular myocardium were detected by Western blot.Cardiac fibroblasts(CFs)were cultured in SD rats by differential adhesion, and CFs were pretreated with different concentrations of SalB(12.5, 25, 50 μmol·L-1)for 1 h, and the optimal dose was determined by high glucose induction.RhoA protein expression in CFs was detected by immunofluorescence; the protein expressions of α-SMA, Collagen , Collagen III, RhoA and ROCK1 in CFs were detected by Western blot.Results Compared with the normal control group, the expression of collagen in left ventricular myocardium of diabetic rats increased significantly, and the expression of α-SMA, Collagen and Collagen III increased significantly.After SalB intervention, the expression of collagen decreased significantly, and the expression of the above proteins decreased significantly(P<0.01).The expression of α-SMA, Collagen , Collagen III, RhoA and ROCK1 in CFs stimulated with 25 mmol·L-1 high glucose for 24 h was significantly increased.After pretreatment with SalB(25 μmol·L-1)and inhibitor Y-27632(10 μmol·L-1), the activity of CFs was significantly inhibited, and the expression of these proteins was significantly decreased(P<0.01).Conclusion SalB can improve myocardial fibrosis in diabetic rats, and has a certain role in protecting the myocardium of diabetic rats.The mechanism may be related to the inhibition of RhoA/ROCK1 signaling pathway.
ABSTRACT
It has previously been reported that 1,25-(OH)2D3 inhibits high glucose-induced epithelial-to-mesenchymal transition (EMT) in HK-2 cells. However, the mechanism of this renoprotective action remains unclear. Elocalcitol (BXL-628), a vitamin D analog, has been suggested to be effective on the RhoA/Rho associated protein kinase (ROCK) pathway, which serves a crucial role in high glucose-induced EMT. The aim of the present study was to investigate the effect of 1,25-(OH)2D3 and its analogue BXL-628 on high glucose-induced activation of the RhoA/ROCK pathway in human renal proximal tubular cells. HK-2 cells were co-treated with high glucose and either 1,25-(OH)2D3 or BXL-628. The RhoA expression levels and ROCK activity of the membrane were assessed via western blot analysis or immunofluorescence. α-smooth muscle actin (α-SMA) and epithelial (E)-cadherin were detected using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), whereas collagen I and fibronectin levels were measured by ELISA and RT-qPCR. The results demonstrated that 1,25-(OH)2D3 and BXL-628 both significantly downregulated the expression of active RhoA and ROCK activity induced by high glucose (P<0.05). Furthermore, the expressions of α-SMA, collagen I, and fibronectin were significantly downregulated at both protein and mRNA (P<0.05) levels, whereas the expression of E-cadherin was significantly increased (P<0.05) by 1,25-(OH)2D3 or BXL-628 treatment. In conclusion, the vitamin D receptor agonist 1,25-(OH)2D3 and its analogue BXL-628 were both able to attenuate high glucose-induced EMT and extracellular matrix accumulation of HK-2 cells by suppressing the RhoA/ROCK signaling pathway in vitro.
