Effects Of Tetramethylpyrazine On Cell Proliferation And The Collagen Synthesis Of Human Embryo Fibroblast Cell Line MRC-5

ObjectiveThe research aimed to investigate effects of different concentrations of TMP on MRC-5cells proliferation through in vitro cell culture, and then observed the changes of expression ofcollagen protein in MRC-5after different concentrations of TMP for different time. Theresults will provide new theory and ideas for traditional Chinese medicine used in preventionand treatment of pulmonary fibrosis.Methods1. Human embryonic lung fibroblast cell line (MRC-5) were cultured and then weretreated with different concentrations of ligustrazine. The cells were divided into five groups:①normal control group,②l igustrazine1×10-3mg/mL group;③l igustrazine1×10-2mg/mLgroup;④ligustrazine1×10-1mg/mL group;⑤ligustrazine1mg/mL group. After cultured24h,48h and72h, the morphological changes of cells were observed under microscope, and thenthe MTT method was used to detect the proliferation of MRC-5after treatment of differentconcentrations of ligustrazine for different time.2. MRC-5cells were divided into:①normal control group,②the dexamethasone group,③ligustrazine1x10-3mg/mL group,④ligustrazine1x10-2mg/mL group,⑤ligustrazine1x10-1mg/mL group,⑥ligustrazine1mg/mL group. After developed in37℃5%CO2incubator for24h,48h and72h, cells were stained with Sirius scarlet and the collagen proteinin cells was observed. Then intracellular protein expression of COLⅠ a nd COL Ⅲwasanalyzed using immunohistochemical method and q RT-PCR was used to detect the mRNAexpression of COL Ⅰ and COL Ⅲ.Results1. After MRC-5cells were treated with different concentrations of ligustrazine for24h,48h and72h, it was found that with the increasing of concentration of the drug and theprolong of the incubation time, rate of cells inhibition increased significantly. Furthermore,under the same treatment interval, rate of cell proliferation inhibition of1mg/mL group ishighest, and under the same concentration, rate of cell proliferation inhibition was mostsignificant after72h treatment. The rate of cell proliferation inhibition of cells increasedgradually over time.2. Sirius scarlet method revealed that with the increase of concentration and the extension of interaction time of ligustrazine, the collagen synthesis was not decreasedsignificantly after treated for24h, while after treated for48h, significant decrease of collagensynthesis was observed, and ligustrazine1mg/mL group exerted most inhibition effects thanother groups as well as dexamethasone group. After incubated for72h, ligustrazine1mg/mLgroup still exhibit strong inhibition effect to collagen synthesis as well as dexamethasonegroup.3. Immunohistochemical method revealed that the expression of COLⅠ protein andCOL Ⅲ protein were suppressed significantly in dexamethasone group, ligustrazine highconcentration group (1mg/ml) and ligustrazine low concentration group (1x10-2mg/ml)compared with normal control group. But the difference in COLⅠ protein and COLⅢprotein expression between dexamethasone group and ligustrazine high concentration group(1mg/ml) was not statistically significant.4. qRT-PCR found that mRNA expression of COLⅠ and COL Ⅲwas significantlyinhibited in dexamethasone group, ligustrazine high concentration group (1mg/ml) andligustrazine low concentration group (1x10-2mg/ml) compared with normal control group,and mRNA expression in ligustrazine high concentration group (1mg/ml) decreased obviouslycompared with ligustrazine low concentration group (1x10-2mg/ml) and dexamethasonegroup.Conclusion:1. Ligustrazine Hydrochloride Injection can inhibit human embryonic lung fibroblast cellline (MRC-5) proliferation and reduce the lung deposition of the cells, which may contributesto the prevention and treatment of pulmonary fibrosis.2. Ligustrazine Hydrochloride Injection has Anti-pulmonary Fibrosis Effect. Themechanism may be associated with inhibiting of the expression of COL ⅠmRNA andCOL ⅢmRNA and protein, and then inhibits the synthesis of intracellular collagen secretion,reduce collagen deposition to the lungs.