Study Of Mechanism Of TRPM7Involved In Proliferation And Differentiation Of Human Lung Fibroblasts

Pulmonary fibrosis represents the end stage of several interstitial lung diseases, and ischaracterized by excessive matrix deposition and destruction of the normal lungarchitecture. Many studies showed that various growth factors, induce cell proliferationand differentiation, which is associated with the pathogenesis of pulmonary fibrosis.Epidemiological found that the morbidity and mortality of pulmonary fibrosis isgradually elevated with increasing age. Recently, The treatment of pulmonary fibrosismainly corticosteroids and immunosuppressive agents, but the effects are not ideal andhave serious side effects. So, Looking for effective drugs for the prevention andtreatment of pulmonary fibrosis, has been our concern.Recent studies also have shown that ion channels play an important role in the processof fibrosis. Transient receptor potential melastain7（TRPM7） channels, members oftransient receptor potential （TRP） channels superfamily, are bifunctional proteins withdual structure of both ion channels and protein kinases, participating in many importantphysical processes such as growth, proliferation, migration and apoptosis. It participatesin the process of fibrosis. However, the presence and potential function of TRPM7channels in the survival of human lung fibroblast are still unknown.The topic is chooses human lung fibroblast （MRC-5） as object, to explore the effect of TRPM7on pulmonary fibrosis. In this research we investigated the role of TRPM7channels in the TGF-β1-induced proliferation of ex-vivo human lung fibroblasts andtheir differentiation into myofibroblasts. This study also was to explore the molecularmechanism of TRPM7on the progression of pulmonary fibrosis. The study includes thefollowing three parts:1. Expression of TRPM7in human lung fibroblast （MRC-5）Through cultured MRC-5in vitro, detect the expression levels of TRPM7in MRC-5using RT-PCR and Western blot. Results suggest that the expressions of TRPM7mRNAand protein were apparently increased by the cultivation of TGF-β1compared to control.2. Inhibition of TRPM7on TGF-β1-induced MRC5cells proliferation anddifferentiationWe investigated the effect of Gd³⁺or2-APB on the proliferation of MRC5cells byMTT assay. Flow cytometry was used to detect cell cycle. Evaluate the levels ofTRPM7, α-SMA, Collagen mRNA by RT-PCR, detect the expression levels of α-SMA,Collagen by Western blot. Results suggest that Gd³⁺or2-APB, non-specific TRPM7channel inhibitor, inhibit the expressions of TRPM7. Gd³⁺or2-APB had a profoundinhibitory effect on TGF-β1-induced proliferation of MRC5cells by promoting theaccumulation of G0/G1period, reducing the number of G2/M and S period. Moreover,Gd³⁺or2-APB could inhibit the expression of α-SMA and Collagen. These resultsshow that the downregulate TRPM7can inhibit the proliferation and differentiation ofMRC-5.Specific TRPM7-siRNA was transfected by Lipofectamine^TM2000to downregulate ofTRPM7in MRC-5. Cell cycle was measured by Flow Cytometry. The expressions ofTRPM7, α-SMA and Collagen were detected by RT-PCR and Western blot. Results suggest that TRPM7-siRNA significantly decreased the proliferation of MRC-5, and theinductions of α-SMA and collagen I were biomarkers of pulmonary fibrosis which wereboth repressed.3. Effects of TRPM7on PI3K/AKT signaling pathway in MRC-5MRC5cells were incubated with TGF-β1,and then treatment with LY294002, which isthe specific blockers of PI3K/AKT signaling pathway, Gd³⁺,2-APB respectively, whichare blockers of ion channels. MTT assay was used to detect the proliferation of MRC-5.The expression levels of P-Akt and Akt by Western blot. The results showed thatTGF-β1could significantly stimulate the proliferation of MRC-5, but after treatmentwith LY294002, the proliferation is inhibited obviously. Compared with the normalgroup, TGF-β1can significantly increase the expression of p-Akt protein. Aftertreatment with LY294002, Gd³⁺and2-APB, the expression of p-Akt protein is inhibitedobviously, but t-Akt protein have no significant change. Furthermore, TRPM7-siRNAsignificantly decreased the the expression of p-Akt protein, but t-Akt protein have nosignificant change.In conclusion, we provide strong evidence that human lung fibroblast express functionalTRPM7channels that play a vital role in modulating cell proliferation anddifferentiation, probably involved in the PI3K/Akt pathway. Based on it, inhibition ofTRPM7channels may prove to be a novel therapeutic approach for pulmonary fibrosis.