高盐对乳鼠心脏成纤维细胞增殖与胶原分泌的影响及相关机制

目的观察高盐对乳鼠心脏成纤维细胞(CFs)增殖及胶原分泌的影响,并分析其可能的相关机制。方法胰酶、Ⅱ型胶原酶两步消化法消化分离乳鼠心肌组织,通过差速贴壁法收集、培养乳鼠CFs。CFs传至第4代去血清同步化24 h,分为对照组(培养基Na+终浓度139 mmol/L)、高盐组(培养基Na^+终浓度146、149、152、155、158、161、164、167、170、173和176 mmol/L),培养24、48、72和96 h后,CCK-8检测CFs增殖情况。选择增殖作用最为明显的Na+浓度和作用时间,以Na^+139 mmol/L为对照进行后续实验,高盐组(培养基Na^+终浓度161 mmol/L),甘露醇组(甘露醇浓度为29.334 mg/ml),其中甘露醇组与高盐组渗透压相同。CCK-8检测渗透压对CFs增殖的影响。酶联免疫吸附试验(ELISA)检测CFs培养上清液中Ⅰ型和Ⅲ型胶原的表达。Western印迹检测CFs中转化生长因子(TGF)-β1、p-smad3及smad7蛋白的表达情况。结果CCK-8结果显示,Na+146~176 mmol/L作用48、72 h可促进乳鼠CFs增殖(Na+161 mmol/L作用48 h最明显);检测渗透压对CFs增殖显示,高盐组(Na+161 mmol/L)细胞发生增殖明显,而甘露醇组细胞较对照组(Na+139 mmol/L)并无增殖差异;ELISA结果显示,高盐组中Ⅰ型和Ⅲ型胶原表达均增加(P<0.05),甘露醇组胶原Ⅰ和胶原Ⅲ表达较对照组比无差异(P>0.05)。Western印迹结果显示,高盐组TGF-β1和p-smad3蛋白表达较对照组和甘露醇组明显增多(P<0.05),smad7蛋白表达明显减少(P<0.05);甘露醇组TGF-β1、p-smad3、smad7蛋白表达与对照组比较无差异(P>0.05)。结论高盐可诱导乳鼠CFs发生增殖,且能够促使Ⅰ型胶原和Ⅲ型胶原分泌增加,且与渗透压改变无关,其机制可能与TGF-β1/smads表达异常相关。

Effects of high sodium on the proliferation and collagen secretion of neonatal rat cardiac fibroblasts cells and related mechanisms

Objective To investigate the effect of high salt on the proliferation and collagen secretion of neonatal rat cardiac fibroblasts cells(CFs),and its possible mechanism.Methods Ventricular tissue was digested with trypsin first and then collagenaseⅡ.Neonatal rat CFs were collected and cultured through the way of differential adhesion.The experimental procedures were performed with the fourth generation of rat CFs.Rat CFs were synchronized by serum starvation for 24 h before high sodium treatment,the cells were cultured in a standard medium containing 139 mmol/L of sodium,the high sodium medium(146,149,152,155,158,161,164,167,170,173 and 176 mmol/L)contained additional sodium chloride,then continually cultured for 24 h,48 h,72 h and 96 h,respectively.The proliferation of CFs was measured using CCK-8 assay.Na^+ concentration and action time were chosen by proliferation effect for follow-up experiment with Na^+ 139 mmol/L as a control and Na+161 mmol/L as high salt group.The osmotic pressure of high salt group was the same as that of mannitol group(mannitol concentration 29.334 mg/ml).The effect of osmotic pressure on the proliferation of CFs was confirmed by CCK-8 assay.ELISA was used to detect the expression of typeⅠcollagen and typeⅢ collagen in the cell culture medium.Western blot was used to detect the expression of transforming growth factor(TGF)-β1,p-smad3 and Smad7 protein in CFs.Results CCK8 assay showed that compared with Na+139 mmol/L group,146~176 mmol/L Na+treatment for 48 h and 72 h could significantly promote the proliferation of CFs,effect of 161 mmol/L Na+was the most obvious.Compared with Na+139 mmol/L group,there was no significant difference in the rat CFs proliferation effect in mannitol group.The results of ELISA showed that the expressions of collagenⅠand collagenⅢincreased in high sodium group(P<0.05),there was no obvious changes of collagenⅠand collagenⅢexpression between mannitol group and control group.Western blot results showed that the expressions of TGF-β1 and p-smad3 protein in high sodium group were higher than those in control group and mannitol group(P<0.05),and the expression of Smad7 protein was decreased(P<0.05),the expressions of TGF-β1,p-smad3 and Smad7 protein in mannitol group was not different from those in control group.Conclusions High sodium could induce the proliferation of rat CFs and increase the collagen secretion.It has nothing to do with the change of osmotic pressure.Its mechanism might be related to the abnormal expression of TGF-β1/Smads.