| Objective:to verify the existence of abnormal oxidative stress and autophagy in the skin lesions of scleroderma model mice,and to observe the regulatory effects of Huoxue Chubi Decoction on oxidative stress,Akt-mTOR autophagy pathway,pro fibrogenic factors and collagen in scleroderma model mice,so as to further explore the therapeutic effect and mechanism of Huoxue Chubi Decoction on scleroderma.Methods:(1)in Experiment 1,60 Balb/c mice were randomly divided into 6 groups:high,medium and low dose TCM groups,positive control group,model group and blank control group,with 10 mice in each group.The blank control group was given subcutaneous injection of sterilized water for injection,and the other groups were given subcutaneous injection of bleomycin 400 μg/ml,0.1ml once a day for 4 weeks.After that,the high,middle and low dose TCM groups were given Huoxue Chubi Decoction with the concentration of 0.46g/ml,0.23g/ml and 0.115g/ml respectively,the positive control group was given colchicine with the concentration of 0.013mg/ml,the model group and the blank control group were given sterilized water for injection by gavage with 0.2ml once a day for 4 weeks.At the end of the 8th week,the mice were sacrificed and skin samples were collected.The content of ROS in skin tissue was detected by fluorescent probe method.The skin thickness and pathological changes were observed by HE staining microscope.The content of collagen was observed by Masson staining microscope.The expression levels of PDGF and TGF-β in skin tissues were detected by immunohistochemistry and Western Blot.The expression sites of PDGF and TGF-β were detected by immunohistochemistry.(2)Experiment 2:After scleroderma modeling and drug intervention in mice,The expression levels of p-Akt,Akt,p-mTOR,mTOR and LC3-Ⅰ/Ⅱ in skin tissues were detected by immunohistochemistry and Western Blot.The expression sites of Akt,mTOR and LC3-Ⅰ/Ⅱ were detected by immunohistochemistry.Results:(1)experiment 1:compared with the blank control group,the weight of mice in the model group decreased significantly(P<0.01);Pathological changes showed obvious thickening of epidermis and dermis,infiltration of inflammatory cells,atrophy of hair follicle and other appendages,obvious reduction of fat layer thickness,thickening and increasing of collagen fibers,structural disorder and deepening of staining.In the model group,the skin thickness,ROS,PDGF and TGF were measured-β Compared with the blank control group(P<0.01),the expression of collagen was significantly increased(P<0.05).After drug intervention,compared with the blank control group:① The weight and skin thickness of positive control group and high-dose group were significantly decreased(P<0.01);The skin thickness decreased in the medium and low dose groups(P<0.05);② the expression of collagen in the positive control group and the high-dose group decreased significantly(P<0.01),and the expression of collagen in the medium and low-dose groups decreased significantly(P<0.05);③ the expression of collagen in the positive control group and the high-dose group decreased significantly ROS levels were significantly decreased in both groups(P<0.01).There was no significant difference between the positive control group and the high-dose group(P>0.05).④ The level of PDGF was significantly decreased in the positive control group(P<0.01),and there was significant difference in the PDGF reduction among the TCM groups(P<0.05).There was no significant difference in the efficacy between the positive control group and the high-dose and medium dose TCM groups(P>0.05),which was better than the low-dose TCM group(P<0.05).⑤The positive control group could significantly down regulate the level of TGF-β(P<0.01),and the curative effect was better than that of the traditional Chinese medicine groups(P<0.05).The levels of TGF-β in high-dose group and low-dose group were decreased(P<0.05).Correlation analysis showed that TGF-β level in skin tissue was strongly correlated with PDGF level(r=0.964,P<0.01)and skin thickness(r=-0.578,P<0.05),PDGF level was strongly correlated with skin thickness(r=0.616,P<0.01);ROS content was strongly correlated with TGF-βlevel(r=0.691,P<0.01),PDGF level(r=0.684,P<0.01)and skin thickness(r=0.635,P<0.01)。(2)Experiment 2:Akt,mTOR activation level and LC3-Ⅰ/Ⅱ expression level of model mice skin were significantly lower than those of blank control group(P<0.01).After drug intervention:①the levels of p-Akt/Akt were significantly up-regulated in each medication group(P<0.01).There was no significant difference between the positive control group and the high-dose group(P>0.05).The effect of the positive control group was significantly better than that of the medium dose group and the low-dose group(P<0.05,P<0.01).The effect of the high-dose group was better than that of the low-dose group(P<0.05).②The level of p-mTOR/mTOR was significantly up-regulated in each treatment group(P<0.01).The effect of high and medium dose groups was better than that of positive control group(P<0.01,P<0.05).The effect of high dose group was better than that of medium and low dose groups(P<0.01,P<0.05).③The expression level of LC3-Ⅰ/Ⅱ in each drug group was significantly up-regulated(P<0.01).There was no significant difference between the positive control group and the high-dose group(P>0.05).The effect of the positive control group was significantly better than that of the medium dose group and the low-dose group(P<0.01).The effect of the high-dose group was better than that of the medium dose group and the low-dose group(P<0.01).The effect of the medium dose group was better than that of the low-dose group(P<0.05))。Correlation analysis showed that ROS concentration was negatively correlated with Akt phosphorylation level(r=-0.610,P<0.01),LC3-Ⅰ/Ⅱ level(r=-0.482,P<0.01),and mTOR level(r=-0.508,P<0.01);Akt phosphorylation was negatively correlated with TGF-β level(r=-0.562,P<0.05)and PDGF level(r=-0.689,P<0.01);mTOR phosphorylation was negatively correlated with PDGF level(r=-0.556,P<0.05)and skin thickness(r=-0.496,P<0.05);Skin LC3-Ⅰ/Ⅱ level was negatively correlated with TGF-β level(r=-0.734,P<0.01),PDGF level(r=-0.843,P<0.01)and skin thickness(r=-0.556,P<0.05).Conclusion:the appearance and pathological changes of skin lesions in scleroderma model mice are consistent with the skin lesions in scleroderma stage.The levels of ROS,PDGF and TGF-β in skin tissue of model mice were significantly increased,and the content of ROS in skin tissue was correlated with the levels of TGF-β,PDGF and skin thickness,which proved that there was abnormal oxidative stress reaction in scleroderma lesions,and there was a certain correlation between oxidative stress and PDGF and TGF-β in the pathogenesis of scleroderma.②The phosphorylation and expression of Akt and mTOR in skin tissue of model mice were significantly decreased,and the expression of autophagy marker LC3-Ⅰ/Ⅱ was decreased,which confirmed the abnormal decrease of autophagy in scleroderma skin.The activity and expression of Akt,mTOR and LC3-Ⅰ/Ⅱ were negatively correlated with TGF-β,PDGF and skin thickness,indicating that autophagy can inhibit the process of fibrosis,and its mechanism may be related to the inhibition of the expression of Pro fibrogenic factors and the degradation of collagen during autophagy.③Correlation analysis showed that ROS was negatively correlated with the activation and expression of Akt,mTOR and LC3-Ⅰ/Ⅱ,suggesting that oxidative stress in scleroderma can inhibit autophagy by regulating the activity of Akt mTOR pathway.④ After treatment with Huoxue Chubi Decoction and colchicine,ROS was inhibited,the activity and expression of autophagy related factors Akt,mTOR and LC3-Ⅰ/Ⅱ were enhanced,and the pro fibrogenic factors TGF-β and PDGF were decreased,which indicated that both of them could promote autophagy by reducing tissue oxidative stress,thus enhancing the degradation ability of collagen,and down regulating Pro fibrogenic factors,thereby reducing skin collagen synthesis.⑤The regulation effect of different dosage groups of traditional Chinese medicine changed with the concentration of the drug,indicating that Huoxue ChuBi decoction may be a dose-dependent drug,and there is a certain threshold of action.In this experiment,the effect of most high-dose application is better. |
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