The Mechanism Of Effect Of Equilibrium Drift Between Relaxin And Endothelin-1on Fibrosis In Systemic Scleroderma

Part I Study of equilibrium drift of relaxin/endotheline-1in peripheral blood of patients with systemic sclerodermaObjective:This study aimed to measure serum levels of relaxin and endothelin-1(ET-1) in patients with systemic scleroderma (SSc) and to infer their relationship to features of SSc.Methods:Fifty-one SSc patients (ages19-75) and51healthy subjects at similar age and gender distribution were enrolled. Patients were classified into two subtypes: diffuse and limited cutaneous SSc. Factors including internal organ involvement, antinuclear antibody (ANA) and anti-Scl-70antibody, were assessed by clinical and laboratory data. Serum relaxin and ET-1were detected by ELISA. Analysis of variance (ANOVA) with Bonferroni’s post-test, multivariate ANOVA (MANOVA) and logistic regression analysis were used for statistical analysis.Results:Both serum ET-1and relaxin were significantly elevated in SSc patients compared to healthy subjects (P<0.001). Moreover, there was a significant difference in the correlation between SSc patients and serum ET-1and relaxin (MANOVA, P<0.001), Logistic regression analysis showed serum relaxin had a higher odds ratio (1.165,95%confidence interval1.097-1.264) than serum ET-1(odds ratio1.061,95%confidence interval1.034-1.095) for predicting SSc. Serum ET-1was increased but serum relaxin was decreased (both P<0.05) in men and postmenopausal women compared to those in fertile women. SSc patients with internal organ involvement had higher serum ET-1than those without internal organ involvement (P<0.001), whereas serum relaxin did not correlate with internal organ involvement. Neither serum ET-1nor relaxin is significantly correlated with the subtypes of SSc, ANA or anti-Scl-70antibody.Conclusion:Both serum ET-1and relaxin, although higher in SSc patients than in healthy subjects, vary with gender and menstrual status. Serum relaxin predicts SSc better than does serum ET-1, whereas elevated serum ET-1indicates more severe SSc. Part Ⅱ Effect of equilibrium drift between relaxin and endothelin-1on proliferation, activation and collagen expression in scleroderma fibroblastsObjective:To investigate effect of equilibrium drift between relaxin and endothelin-1on proliferation, activation and collagen expression in scleroderma fibroblasts.Methods:Fibroblast cell lines derived from sclerotic skin of SSc patients and skin of normal controls were established. Fibroblasts of Passage3-6were used in the following tests. The fibroblasts were treated with ET-1(0.25mg/L) alone or in combination with relaxin (1,10and100μg/L). Cell proliferation was measured by MTT assay. Expression of a-smooth muscle actin (a-SMA) was detected by immunohistochemistry. al(Ⅰ) procollagen (COL1A1), COL3A1, matrix metalloproteinase(MMP)-1/tissue inhibitors of MMP (TIMP)-l mRNA levels were measured by real-time PCR.Results:ET-1(0.25mg/L) increased the proliferation of both SSc and normal fibroblasts (P<0.05or P<0.01), while relaxin (1,10and100μg/L) alleviated the stimulatory effect of ET-1on the proliferation of fibroblasts (P<0.05or P<0.01) in a dose-dependent manner. Relaxin (1,10and100μg/L) also alleviated the stimulatory effect of ET-1(0.25mg/L) on expression of a-SMA and COL1A1, COL3A1and TIMP1mRNA levels (P<0.05or P<0.01) and the inhibitory effect of ET-1on MMP1mRNA level (P<0.05or P<0.01) of SSc and normal fibroblasts, in a dose-dependent manner.Conclusion:In vitro, ET-1promotes the proliferation, activation and expression of collagen in SSc fibroblasts. RLN alleviates the above effects of ET-1in a dose-dependent manner. Part III Study on mechanism of correlation of relaxin/endothelin-1with fibrosis in bleomycin induced scleroderm miceObjective:To investigate mechanism of correlation of relaxin/endothelin-1with fibrosis in bleomycin (BLM) induced scleroderm mice.Methods:An established model of skin and lung fibrosis was used, in which6-wk-old female C3H/He mice were administered BLM via injection subcutaneously daily for3weeks. Mice were dosed, via subcutaneously implanted microosmotic pumps, with either vehicle or relaxin (0.5mg/kg/d) for14days beginning on the first day of BLM administration. The dermal thickness and lung fibrosis were assessed by hematoxylin-eosin and Masson-trichrome staining. At different endpoints, plasma level of ET-1was detected by ELISA and mRNA levels of ET-1and COL1A1of skin and lung were measured by real-time PCR.Results:Dermal thickness and lung fibrosis of BLM-injected sclerotic mice were higher than PBS-injectd mice(P<0.05, P<0.001). Compared to PBS-injectd mice, COL1A1mRNA level of skin and lung in BLM-injected mice increased with time (P<0.05, P<0.01). Dermal thickness and lung fibrosis of BLM+RLN-treated mice were lower than those of BLM+NaAc-treated mice (P<0.05, P<0.01). Compared to BLM+NaAc-treated mice, COL1A1mRNA level of skin and lung in BLM+RLN treated mice decreased with time (P<0.05, P<0.01). At the end of W1, plasma ET-1levels of BLM-injected mice and BLM+NaAc-treated mice were higher than PBS-injectd mice and BLM+RLN treated mice, respectively (both P<0.01). Afterward, there were no diference between them. ET-1mRNA level of skin and lung in BLM-injected mice was higher than that of PBS-injectd mice at the end of W1(P<0.01). ET-1mRNA level of skin of BLM+RLN-treated mice was significantly lower than that of BLM+NaAc mice (P<0.05). Afterward, there were no diference between them. ET-1mRNA level of lung of BLM+RLN-treated mice was lower than BLM+NaAc-treated mice (P<0.01, P<0.05).Conclusion:Relaxin induces ET-1reduction in plasma and fibrotic tissues in BLM induced sclerotic mice especially at early phase, which may be significantly involved in the antifibrotic effect of relaxin.