According to modern medicine, RIF is characterized by degeneration and loss of renal tubule and accumulation of extracellular matrix, which is involved with TGF-β1, angiotensin II, HER2 and other biological factors. As a fibrogenic factor, TGF-β1 can induce the activation of myofibroblasts and inhibit the degradation of collagen fibers, which resulting in the accumulation of large amounts of collagen fibers between tissues[15]. Studies have shown that TGF-β1 plays an initial activation role in the formation of fibrosis. TGF-β1 can bind to receptors on the cell surface, activate multiple cell signaling pathways, and induce the expression of target genes, thus promoting the generation of fibrosis [16].
TGF-β1 has been shown to rapidly activate PI3 kinase in a variety of cellular systems, leading to activation of Akt kinase [17].Akt is a central regulator of multiple pathways involved in cell survival, cell size control, and cell migration. Akt regulates cell functions by phosphorylating a variety of downstream factors such as enzymes, kinases and transcription factors. Inhibitors of PI3 kinase and Akt, or a dominant negative form of Akt, have been found to inhibit TGF-β1-induced EMT[18]. mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell movement, protein synthesis and transcription. Over-activated mTOR promotes EMT, and MTOR inhibitors inhibit bleomycin-induced pulmonary fibrosis and EMT in mice [19].
The EMT changes of RTECs are closely related to the damage of gap junction intercellular communication(GJIC). GJIC mediated by Cx43 is involved in the transmission of energy and information between cells, and plays an important role in the regulation of cell proliferation, differentiation, metabolism and internal environment stability and other physiological processes [20]. Impaired intercellular information transmission can lead to decreased cell proliferation, cell cycle arrest and cell hypertrophy, and then increase secretion of extracellular matrix, which eventually leads to the occurrence of renal interstitial. The phosphorylation of CX43 directly affects the assembly, degradation of CX43 on the cell membrane and the GJIC mediated by gap junctions. There are at least five serine phosphorylation sites in connexin 43, which are necessary for the aggregation and intercellular junction of connexin 43. It has been proved that there are action sites of protein kinase C and mitogen activated protein kinase on the molecule of connexin 43, which are related to the closure of gap junction channels [21].
James CC et al. [22] proved that during the process of cell transdifferentiation, the reduction of Cx43 protein can act as a key link to activate PI3K/ Akt /mTOR to further promote the occurrence of cell transdifferentiation. Studies have shown that the glomerular mesangial cells exposed to high concentration of glucose could reduce CX43, inhibit PTEN, trigger Akt phosphorylation, activate downstream mTOR, and lead to GMC hypertrophy [23].On the contrary, overexpression of CX43 could inactivate PTEN/PI3K/Akt/M-TOR pathway, prevent Akt and mTOR phosphorylation, and reverse GMC hypertrophy [23]. Kuang Jy et al. [24] believed that the negative correlation between CX43 expression and Akt/ ERK activation was caused by the direct interaction between Akt/ ERK and CX43. They found that the T286/A305/Q308/Y313 residues and S272/S273 at the carboxyl terminus of CX43 were crucial for its binding to Akt and ERK, respectively. In this study, Western Blot, immunofluorescence and other studies confirmed that up-regulation of CX43 could inhibit the TGF-β1-induced EMT process in RTECs. We further observed that when lentivirus shRNA-CX43 was used to decrease the expression of CX43, the phosphorylation levels and gene expressions of Akt and mTOR were significantly increased, whereas when lentivirus OE-CX43 was used to increase the expression, the phosphorylation levels and gene expressions of Akt and mTOR were significantly decreased. All these indicate that Akt/mTOR signaling pathway may be the downstream molecular mechanism of CX43 regulating EMT. ASV has specific inhibitory effects on cell transdifferentiation and tissue fibrosis, and its mechanism involves complex signaling pathways.
Studies have shown that ASV inhibits PI3K/Akt/mTOR signal and prevents epithelial mesenchymal transdifferentiation (EMT) process by down-regulating TGF-β1 expression [25].In this study, different concentrations of ASV were used to intervene TGF-β1-induced Rtecs cell transdifferentiation. Compared with the model group, ASV could reduce the expression of a-SMA, vimentin and other genes and proteins, and enhance the expression of E-cadherin molecule, indicating that ASV had anti-transdifferentiation effect, which was consistent with the existing studies. After drug intervention, the expression level of CX43 was increased and the phosphorylation level of PI3K/ Akt /mTOR was decreased in a dose-dependent manner. Combined with existing studies, it was speculated that the inhibition of TGF-β1-induced RTECs cell transdifferentiation by ASV might be related to the inhibition of phosphorylation of Akt and mTOR by regulating the expression of CX43 molecule.