[The effect of hypoxia on the expression of RHO/RHO signaling pathway and CTGF of human embryonic fibroblasts].

OBJECTIVE

To observe the effects of hypoxia on cell proliferation and the expression of RHO/RHO signaling pathway, connective tissue growth factor (CTGF) and the collagen type I (Col I) in human embryonic fibroblasts at different time points, and study the possible mechanism of hypoxia-induced pulmonary fibrosis.

METHODS

Human embryonic fibroblasts were cultured under hypoxic condition (37 degrees C, 5 CO2, 2% O2, 93% N2) for 0 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h and 60 h respectively. The protein levels of HIF-1alpha, RhoA, ROCKland CTGF were assayed by Western blot. RT-PCR was performed to detect the mRNA levels of HIF-1alpha, ROCK1, CTGF and Col I. The concentration of collagen type I (Col I) in fibroblast cells supernatant were determined by ELISA.

RESULTS

(1) Basal levels of RhoA, ROCK1, CTGF protein of MRC-5 were observed in normoxia, but the protein levels of RhoA, ROCK1 and CTGF were up-regulated after 1.5 h of hypoxia and increased further with longer exposure to hypoxia. It also demonstrated significant positive correlation between the protein levels of RhoA, ROCK1 and CTGF at different time points of hypoxia. (2) After 1.5 h, mRNA expression of ROCK1, CTGF and Col I was elevated and increased further with longer exposure to hypoxia and peaked after 60 h of hypoxia compared to normoxia. (3) Compared with normoxia, the concentration of Col I in fibroblast cells supernatant increased in different time points of hypoxia and peaked after 24 h of hypoxia.

CONCLUSION

Different time points of hypoxia all significantly up-regulated the expression of protein and mRNA of CTGF and increased the secretion of Col I. Hypoxia could represent a potential brotic stimulus possibly through activation of the RHO/RHO signaling pathway, up-regulating the expression of HIF-1alpha and CTGF, triggering a series of brotic responses.