The Proliferation Of Human Skin Fibroblasts Cultured In Vitro With Traditional Chinese Medicine For Invigorating Qi And Blood And The Effect Of Growth Factor Expression

BackgroundChronic skin ulcer is a common surgical disease with a long course,which affects the quality of life of patients and brings heavy burden to the society.The traditional Chinese medicine treatment for skin ulcer is the method of removing putrescence and generating muscle.Facing the clinical problem of removing putrescence and producing muscle without putrescence,professor tang hanjun innovatively put forward the treatment method of removing blood stasis and generating muscle and replenishing deficiency and generating muscle with remarkable clinical effect.Tonifying qi and blood is an important method for tonifying asthenic muscle.Qi can give birth to blood,and blood can nourish qi.Wound healing is closely related to body qi and blood.Western medicine believes that wound healing is a continuous,complex and coordinated process,and the formation of granulation tissue is the key to wound healing.Granulation tissue is mainly composed of a large number of fibroblasts and capillaries.Proliferation of fibroblasts and growth factors EGF,TGF-β and VEGF secreted by fibroblasts are key factors in wound repair.In previous animal experiments,it was found that traditional Chinese medicine for invigorating qi and blood could promote the proliferation of fibroblasts in rat back wound granulation tissue,up-regulate the synthesis and secretion of vascular endothelial growth factor,accelerate the formation of wound microcapillaries,and accelerate the growth of wound granulation tissue,thus promoting wound healing in rats.According to the "qi and blood" theory of traditional Chinese medicine and the basis of previous animal experiment research,this experiment studied the effect of supplementing qi and blood traditional Chinese medicine on the proliferation and synthesis and secretion of growth factors EGF,TGF-β and VEGF of human skin fibroblasts cultured in vitro.To further explore the mechanism of TCM in promoting wound healing,enrich the basic research content of TCM in promoting wound healing,and lay a foundation for the research on internal medicine for chronic skin ulcer.ObjecticsIn this experiment at the early stage of the TCM theory of "qi" and animal experimental study foundation,further observation of qi and blood tonic medicine(sijunzi decoction,siwu soup,eight Jane soup)on human skin fibroblast cell cycle,cell proliferation,the influence of the human skin fibroblasts synthesized secrete growth factor EGF,TGF-β,VEGF,on the effects of human skin fibroblasts EGF m RNA,TGF-β m RNA,VEGF m RNA.MethodsHuman skin fibroblasts were resuscitated,subcultured and cultured.The Cell Counting kit-8(CCK8)method was used to determine the effect of traditional Chinese medicine(TCM)on the proliferation of human skin fibroblasts in the culture environment of sijunzi tang,siwu tang and bazhen tang for the 4th to 6th generation cells at different points in time.Flow cytometry(FCM)was used to detect the effect of traditional Chinese medicine on the proliferation cycle of human skin fibroblasts.Western blot assay(Wb)was used to detect the effect of traditional Chinese medicine on the synthesis and secretion of EGF,TGF-β1 and VEGFA in human skin fibroblasts.Quantitative real-time polymerase chain reaction(rt-pcr)was used to determine the effect of traditional Chinese medicine on EGF m RNA,TGF-β m RNA and VEGF m RNA of human skin fibroblasts.Results1.Sijunzi decoction(1mg/L～1g/L)interfered in human skin fibroblasts for 24 h.Compared with the control group,sijunzi decoction(10mg/L,100mg/L)can promote cell proliferation and the number of cell growth is different(P<0.05),and sijunzi decoction(100mg/L)can promote cell proliferation more significantly(P<0.01).Sijunzi decoction(1mg/L～1g/L)interfered with human skin fibroblasts for 48 h,and no significant difference was found in the number of cell growth compared with the control group(P>0.05).Siwu decoction(10ug/L～1g/L)interfered in human skin fibroblasts for 24 h.Compared with the control group,siwu decoction(10ug/L～1g/L)could promote cell roliferation and the number of cell growth was different(P<0.05),and siwu decoction(1mg/L～100mg/L)could promote cell proliferation more significantly(P<0.01).Siwu decoction(10ug/L～1g/L)intervened human skin fibroblasts for 48 h.Compared with the control group,siwu decoction(1g/L)could promote cell proliferation,and the number of cell growth was different(P<0.05),while siwu decoction(10ug/L～100mg/L)had nosignificant difference in the number of cell growth(P>0.05).Bazen decoction(10ug/L～1g/L)intervened human skin fibroblasts for 24 h.Compared with the control group,bazen decoction(10ug/L～1g/L)could promote cell proliferation and the number of cell growth was different(P<0.05),and bazen decoction(1mg/L～1g/L)could promote cell proliferation more significantly(P< 0.01).Bazen decoction(10ug/L～1g/L)intervened human skin fibroblasts for 48 h,and there was no significant difference in cell growth number between the two groups(P>0.05).2.Compared with the control group,the proportion of skin fibroblasts at G0/G1 phase was decreased in the sijunzi decoction group,siwu decoction group and bazhen decoction group,and the cell proliferation index PI was increased,showing a significant difference(P<0.01).The proportion of skin fibroblasts in S phase increased significantly in sijunzi decoction group,siwu decoction group and bazhen decoction group(P < 0.01).Compared with sijunzi decoction group,siwu decoction group of human skin fibroblasts G0/G1 phase ratio is reduced,increased cell proliferation index(PI,obvious difference(P<0.05),and human skin fibroblasts S phase proportion reduce no obvious difference(P>0.05),8 soup group of human skin fibroblasts G0/G1 phase proportion reduced more significantly(P<0.01),human skin fibroblasts S phase proportion reduced obviously(P<0.01).Compared with the siwu decoction group,the proportion of skin fibroblasts in the G0/G1 phase was decreased in the bazengtang group,and the proliferation index PI was increased,with a significant difference(P<0.01).Meanwhile,the proportion of skin fibroblasts in the S phase was significantly increased in the bazengtang group(P < 0.01).3.Compared with the control group,EGF expression was significantly increased in the sijunzi decoction group,siwu decoction group and bazhen decoction group(P<0.01).Compared with the sijunzi decoction group,the expression of EGF in the siwu decoction group was decreased(P<0.05),and that in the bazhen decoction group was significantly decreased(P<0.01).Compared with siwu decoction group,EGF expression was decreased in ba zhen decoction group(P<0.05).4.Compared with the control group,the expression of TGF-β1 was significantly increased in the sijunzi decoction group,siwu decoction group and bazhen decoction group(P<0.01).Compared with sijunzi decoction group,there was no significant difference in the expression of TGF-β1 in siwu decoction group(P>0.05),and theexpression of TGF-β1 was increased in ba zhen decoction group(P<0.05).Compared with the siwu decoction group,the expression of TGF-β1 increased in the ba zhen decoction group(P<0.05).Compared with the control group,VEGFA expression increased in the sijunzi decoction group and bazhen decoction group(P<0.05),and significantly increased in the siwu decoction group(P<0.01).Compared with sijunzi decoction group,there was no significant difference in VEGFA expression between siwu decoction group and bazhen decoction group(P>0.05).Compared with siwu decoction group,VEGFA expression in bazhen decoction group was not significantly different(P>0.05).Compared with the control group,the EGF expression increased in the ba zhen tang(100mg/L)group(P<0.05),the EGF expression increased in the ba zhen tang(10mg/L)group was not significantly higher(P>0.05),and the EGF expression decreased in the ba zhen tang(1g/L)group(P<0.05).Compared with the control group,the expression of TGF-β1 in the ba zhen decoction(100mg/L)group was significantly increased(P<0.01),the expression of TGF-β1 in the ba zhen tang(10mg/L)group was not significantly increased(P>0.05),and the expression of TGF-β1 in the ba zhen decoction(1g/L)group was decreased(P<0.05).Compared with the control group,the expression of VEGFA increased in the bazhen decoction(10mg/L)group(P<0.05),significantly increased in the bazhen decoction(100mg/L)group(P<0.01),and decreased in the bazhen decoction(1g/L)group(P > 0.05).5.Compared with the control group,EGFm RNA expression was significantly increased in the sijunzi decoction group and siwu decoction group(P<0.01).EGF m RNA expression increased in the ba zhen tang group(P<0.05).Compared with sijunzi decoction group,siwu decoction group EGF m RNA expression was significantly decreased in the zhentang group(P<0.01).Compared with siwu decoction group,ba zhen decoction group EGF m RNA expression was significantly decreased(P<0.01).Compared with the control group,the m RNA expression levels of TGF-β in sijunzi decoction group,siwu decoction group and bazhen decoction group were significantly higher Height increased(P<0.01).Compared with sijunzi decoction group,the increase of TGF-β m RNA in siwu decoction group was not obvious(P>0.05),the expression level of TGF-β m RNA in the ba zhen tang group was significantly increased(P<0.01).Compared with siwu soup group The expression level of TGF-β m RNA was significantly increased(P<0.01).Compared with the control group,m RNA levels of VEGF in sijunzi decoctiongroup,siwu decoction group and bazhen decoction group were significantly higher Height increased(P<0.01).Compared with sijunzi decoction,VEGF m RNA expression in siwu decoction group was significantly increased(P<0.01),the m RNA expression of VEGF in the bazhen decoction group was significantly decreased(P<0.01).Compared with siwu soup group The m RNA expression of VEGF was significantly lower in the bazhen decoction group than in the control group(P<0.01).Conclusions1.Sijunzi decoction,siwu decoction and bazhen decoction can accelerate the mitosis of human skin fibroblasts and promote the proliferation of human skin fibroblasts.When the concentrations are all 100mg/L,the effect of promoting cell proliferation is obvious and the effect of drug intervention on cell proliferation is obvious after 24h.2.Chinese herbal medicines for invigorating qi and blood promote the secretion of EGF,TGF-β1 and VEGFA by up-regulating the expression of EGF m RNA,TGF-βm RNA and VEGF m RNA in human skin fibroblasts.3.There were differences in the secretion and expression levels of EGF,TGF-β1,and VEGFA promoted by traditional Chinese medicine for invigorating qi and blood.EGF,TGF-β1,and VEGFA played different roles in different stages of wound repair.4.Traditional Chinese medicine for invigorating qi and blood may accelerate the proliferation of human fibroblasts,accelerate angiogenesis,promote the growth of granulation tissue and epithelialization of wound surface,and ultimately promote wound healing.5.bazhen decoction concentration(100mg/L)set of EGF can promote the synthesis of human skin fibroblasts secretion,TGF-β1,VEGFA,bazhen decoction low concentration group(10mg/L)promote the synthesis of human skin fibroblasts secretion of EGF,TGF-β1 effect is not obvious,and bazhen decoction high concentration group(1g/L)for human skin fibroblasts synthetic secretion of EGF,TGF-β,VEGFA appear different degree of inhibition.Reasonable concentration should be used in clinical application of traditional Chinese medicine for invigorating qi and blood.