| Objective:Normal wound healing is divided into three overlapping phases, namely inflammation, proliferation and remodeling, and ends with scar. HS occurs in trauma after one month, and it is is caused by severe trauma, in-depth burning, inflammatory reaction,not only affects the appearance, and easily lead to various limbs.. At present, scar pathogenesis is not fully understood.There are numerous clinical treatments of HS, but with ineffective efficacy. Qin chuan compoundary are formed by traditional Chinese medicine extract components of Ligusticum chuanxiong Hort) (ferulic acid sodium), sinomenine formed in a 2:3 ratio configuration, and two kinds of extract ingredients have anti-inflammatory and analgesic pharmacological effects. Recently it is found that SF and anti liver, lung and kidney fibrosis. sinomenine can also inhibit renal interstitial fibrosis. In addition, the previous studies on animal model of rabbit scar, SF, sinomenine by 2:3 proportion of the configuration of the complexes can effectively inhibit the scar hyperplasia of rabbit. Through in-vitro experiment, to study Qin chuan compoundary'impact on the fibroblasts (HSFB) proliferation of human hyperplastic scar, apoptosis and the synthesis of collagen and TGF beta/Smad Protein signal transduction pathway in multiple related signal protein content. To clarify the biological effect of Qingchuan complex on HSFb and to explore its possible mechanism from the signal transduction pathway, and to provide experimental evidence for clinical application of HS.Method:1.Identification and generation cultivation of HSFb3-4 cell line HSFb cell are purchased from Shanghai Sur Biotechnology Limited Cell Bank. The cells were identified by HE, immunocytochemistry staining and it's confirmed that the cells were hypertrophic scar fibroblasts. Medium containing 10% fetal bovine serum DMEM,37?,5%CO2, are cultivated and generated under saturated humidity, and this experiment adopts 4-6 generation cells.2.Experimental group and MTT method were used to determine the LC50 and Optimal effective timeTo establish three medicine experiment groups, including the negative control group, Kawa So group, sinomenine group, and Chuanqing group; adopting MTT method to determine the effective dose and optimal time for the three drug groups on HSFB IC50 when they are at different levels, high, medium and low.3.MTT effects on HSFb's reproduction.MTT method was used to detect the effects of different drugs on cell proliferation.4.Impact on the morphology, proliferation and apoptosis of HSFbElectron microscope is used to observe the effects of each group of drug on the morphologh of the cells; TUNEL method is employed to detect the effect on cell apoptosis; immune histochemistry is used to test the impact on apoptosis related to Bcl-2, Bax, caspase-3 expression.5.Effect on HSFb collagen systhesisAfter drug of different concentrations working on HSFb, effect of detection of Western blot method for the synthesis of type ? and ? collagen.6.Effect on the signal pathway of HSFb TGF-?/SmadAfter drug of different concentrations working on HSFb. Western blot is used to test the effect on protein TGF-?1, p-Smad2/3,p-Smad4 and p-Smad7, which are the important signal molecule of TGF-?/Smads signal pathway. To test and compare signal molecule like TGF-?1 p-Smad2/3, p-Smad4 and p-Smad7, and their influence on the reproduction, apoptosis and collagen synthesis of HSFb.Result:1.HE and immunohistochemical identification purchased cell line of human hypertrophic scar fibroblast.2.SF, sinomenine and Qin chuan compoundary HSFb's LC50 are 0.3075mg/ml, 1.1285 mg/ml, and 0.938mg/ml respectively;the optimal time for each group is 48h by MTT;SF doses are 0.003mg/ml,0.03mg/ml,0.3mg/ml; Qin chuan compoundary high, medium and low level doses are 0.008 mg/ml,0.08 mg/ml,0.8mg/ml.3.MTT shows that, except the low concentration of Qingchuan compoundary group, the rest groups of drugs can inhibit cell proliferation in varying degrees.4. SF, sinomenine and Qin chuan compoundary working on HSFb may lead to HSFb's microstructure change:Such as the cell volume was smaller, irregular shape, nuclear staining matter accumulation or nucleolar enlargement, reduction of rough endoplasmic reticulum and cytoplasm appeared vacuoles or apoptotic bodies TUNEL shows that, except the low concentration of sinomenine group, the rest groups of drugs can induce cell apoptosis rate of drug group increased, immune histochemistry showed that sinomenine (high,middle) group. Qingchuan compoundary (high) and SF (high) groups can decrease Bcl-2 protein expression;, high and middle concentrations of each drug can increase regulation of Bax and caspase-3 protein.5. SF, sinomenine and Qin chuan compoundary working on HSFb, Western blot shows that, except the low concentration of SF group with col alpha 1 (?) protein expression had no inhibitory effect on the outside, the rest groups of drugs can explicitly inhibit the Cok?1(?), Col?1(?) expression.6. Western blot shows that, SF(high,middle) group,sinomenine (highH) group and Qingchuan compoundary (high) group can inhibit TGF-beta 1 expression of HSFB;(2) SF(high,middle) group, sinomenine (high) group and Qingchuan compoundary (high, middle) group can inhibit p-Smad2/3 expression of HSFB,and it's inhibition of strength with the concentration increase and enhance;(3)SF(high,middle) group, sinomenine (high) group and Qingchuan compoundary (high,middle) group can inhibit p-Smad4 expression of HSFB;(4)SF(high,middle) group, sinomenine (high)group and Qingchuan compoundary (high) group can up p-Smad7 expression.Qin chuan compoundary (high), SF(high,middle), and sinomenine(high) group in the inhibition of cell TGF beta-1 expression.at the same time, can significantly inhibit HSFB proliferation and induced significant apoptosis and inhibit the col alpha 1 (?), col alpha 1 (?) protein expression.Qin chuan compoundary (high, medium), SF(high,middle), and sinomenine(high) group in the inhibition of p-Smad2/3, P-Smad4 expression,,at the same time, were to inhibition of col alpha 1 (?), col alpha 1 (?) protein expression.Qin chuan compoundary (high), SF(high.middle) and sinomenine(high) raised P-Smad7 content, at the same time, can induce cell apoptosis and inhibit the col alpha 1 (?), col alpha 1 (?) protein expression.Conclusion:1.Qin chuan compoundary from the concentration of 0.08 mg/ml can effectively inhibit the proliferation of HSFb, the intensity of the inhibition increased with the increase of concentration;2.Qingchuan compoundary can affect Ultrastructural morphologyof HSFB and induce cell apoptosis; Qingchuan compoundary can decrease Bcl-2 protein expression, increase Bax and caspase-3 protein expression,it maybe the molecular basis of HSFB cells apoptosis.3.Qingchuan compoundary can affect the biological function of HSFb, including the inhibition of cell of type ? and ? collagen synthesis, reduce the secretion of TGF-beta 1 protein4.Qin chuan compoundary may inhibit TGF-?1 proliferation, p-Smad2/3, p-Smad4 protein expression and raised the p-Smad7 protein,so as to hinder the TGF beta/ Smad signaling pathway, then inhibit HSFB proliferation, collagen synthesis and induction of apoptosis. |
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