Mechanism Of Huacai Xuefu Zhuyu Decoction In The Treatment Of Lower Extremity Arteriosclerosis Obliterans

Objective:To analyze the characteristics of Professor Zhao Gang’s traditional Chinese medicine prescription in the treatment of ASO through data mining,To extract the basic core of the treatment of this disease,prescription of cutting Huacai Xuefu Zhuyu Decoction;observe the efficacy and safety of HCXFZY Decoction in the clinical treatment of Fontaine(Ⅰ-Ⅲ)ASO;The possible mechanism of action was preliminarily explored through cell experiments,which provided theoretical basis for its clinical promotionMethod:Part 1:From September 2010 to September 2020,388 Cases of ASO patients in the Department of peripheral vascular diseases,the First Affiliated Hospital of Heilongjiang University of traditional Chinese medicine were selected as the research objects,and the database was established.The traditional Chinese Medicine Inheritance computing platform(V3.0)was used to carry out frequency statistics,association rule analysis and cluster analysis on the basic information,syndrome types and prescription drugs of patients,and the data mining results were analyzed in depth.Part 2:A total of 45 ASO patients admitted to the Department of peripheral vascular diseases,the First Affiliated Hospital of Heilongjiang University of traditional Chinese medicine from September 2020 to December 2020 were included,including 3 patients in the treatment group,treated with huacaixuefuzhuyu decoction combined with beraprost sodium tablets.22 patients in the control group,treated with oral beraprost sodium tablets.XFZY decoction consists of Taoren 20g,Honghua 20g,Chishao 15g,Chuanxiong 15g,Niuxi 15g,Danggui 20g,Shudihuang 20g,dangshen 20g,Huangqi 20g and Zhigancao 15g.The decoction is prepared by the First Affiliated Hospital of Heilongjiang University of traditional Chinese medicine,200 ml in the morning and 200 ml in the evening,with a course of 4 weeks.The TCM symptom scores and clinical efficacy of the treatment group and the control group before and after treatment were compared,ABI index,infrared thermography,TG、TC、HDL-C、LDL-C、hs-CRPand FIB of the treatment group and the control group before and after treatment were compared,and SPSS 23.0 software was used for statistical description and analysis,P<0.05.Part 3:Rat vascular smooth muscle cells(VSMCs)were cultured in vitro,and the injury model of VSMCs was induced by ox-LDL.The experiment was divided into 6 groups:blank group,model group,low dose group,medium dose group,high dose group and positive drug group.Cell morphology was observed by inverted phase contrast microscope.BrdU staining,Transwell cell migration test and scratch test were used to detect the proliferation and migration of VSMCs The protein expression levels of α-SMA,CRBP1,PCNA,MMP-9,TGF-β1,TpRI,p-Smad3 and t-Smad3 were detected by blot;the expression levels of α-SMA and CRBP1 were detected by immunofluorescen-ce;the expression levels of p-Smad3 were observed by laser confocal microscope;the mRNA expressions of MMP-9 and PCNA in VSMCs were detected by RT-PCR.Result:First part:1.The number of patients with ASO in different age groups and gender frequency statistics:29 patients were 40-49 years old,63 patients were 50-59 years old,133 patients were 60-69 years old,123 patients were 70-79 years old,and 40 patients were over 80 years old.There were 244 males and 144 females in 388 ASO patients.2.Statistics of TCM syndrome type frequency:according to the ascending and descending order of frequency,there were 139 cases of blood stasis syndrome,67 cases of cold coagulation and blood stasis syndrome,47 cases of qi stagnation and blood stasis syndrome,29 cases of blood deficiency and blood stasis syndrome,27 cases of yin deficiency and blood stasis syndrome,22 cases of cold dampness and blood stasis syndrome,16 cases of phlegm and blood stasis syndrome,13 cases of Qi and blood deficiency syndrome,13 cases of damp heat and blood stasis syndrome,11 cases of Yang deficiency and blood stasis syndrome,4 cases of yin deficiency and blood stasis syndrome.3.Three Statistics of drug use frequency:a total of 184 kinds of drugs were used,the sum of the use frequency of all drugs was 6310 times,and 53 kinds of drugs with drug use frequency≥ 30;among them,17 kinds of drugs with drug use frequency≥ 100 were ranked according to the ascending and descending order,which were Danggui,Niuxi,Shudihuang,Chishao,Taoren,Huangqi,Dilong,Honghua,Zhigancao,chuanxiong,Shengdihuang,Chaihu,Chaobaizhu,Dangshen,Fuling,Guizhi;Zhiqiao.4.Statistics of four Qi,five flavors and meridians of drugs:the frequency of four Qi of drugs was statistically analyzed:the frequency of use of warm drugs was 2277,cold 1678 times,Pingyi 1607 times,and thermal 151 times.The frequency of five flavors of the drugs was statistically analyzed:3072,2721 bitter,1830,800 acid,494 salty ones.The frequency of drug channel was statistically analyzed:liver meridians were 3748 times,spleen meridian 2435 times,heart meridian 2323 times,kidney meridian 1812 times,and lung meridian 1709 times.5.Medication pattern statistics:the number of support was set at 130,and the confidence level was 0.9.The results showed that there were 198 commonly used combinations of traditional Chinese medicine,such as"Danggui,Niuxi";Four kinds of medicine combinations are commonly used,such as "Danggui,Niuxi,Taoren,Honghua";five kinds of medicine combinations are commonly used,such as "Danggui,Niuxi,Shudihuang,Taoren,Honghua".6.Rule analysis and Statistics:set the number of support at 130 and the confidence level at 0.9 for rule analysis.Whenever the left drug is ">",the probability of right drug appearing in 388 prescriptions is more than 99%,and 134 rules are obtained through platform system analysis.7.Cluster analysis results:set the number of cluster points to 5,and extract 5 core TCM combinations,including 104 frequency statistics of core 1,89 frequency statistics of core 2,77 frequency statistics of core 3,61 frequency statistics of core 4 and 57 frequency statistics of core 5.The second part:1.Main outcome measures:the scores of TCM symptoms of patients with arteriosclerosis obliterans of lower limbs were compared within the group,and the scores after treatment were better than those before treatment(P<0.05);the results between the groups showed that the improvement degree of the treatment group was better than that of the control group(P<0.05).2.Secondary outcome measures:the ankle brachial index and lower extremity infrared thermography temperature were significantly better after treatment than before treatment(P<0.05);the results of comparison between groups showed that the treatment group was better than the control group(P<0.05).Compared with before treatment,the laboratory indexes of TG,TC,LDL-C,hs-CRP and FIB in the treatment group were significantly decreased,while HDL-C was significantly increased,which was significantly better than before treatment(P<0.05);compared with before treatment,the improvement of laboratory indexes in the treatment group was better than that in the control group(P<0.05).3.Clinical efficacy evaluation of the two groups:22 cases were markedly effective and 1 case was effective in the treatment group;1 case was clinically cured,6 cases were markedly effective,14 cases were effective and 1 case was deteriorated in the control group,the difference was statistically significant(P<0.05).The third part:1.Observation of cell morphology:the morphology of VSMCs in blank group was normal,showing typical "peak valley" structure.After ox-LDL treatment,the number of cells decreased,the morphology was irregular,and the pseudopodia retracted obviously.With the increase of huacaixuefuzhuyu Decoction concentration,VSMCs appeared peak valley structure.2.Cell proliferation test results showed that:compared with the blank group,the proliferation activity of VSMCs in the model group was significantly enhanced;HCXFZY decoction had a concentration dependent positive correlation with inhibition of proliferation activity(P<0.05),3.The results of smooth muscle cell migration experiment showed that:compared with the blank group,the migration ability of VSMCs gradually increased,and the migration ability of VSMCs gradually decreased with the increase of the concentration of HCXFZY decoction(P<0.05).4.The expression levels of α-SMA,CRBP1,PCNA,MMP-9 and TGF-β1/Smad3 pathway related proteins were detected by Western blot.Compared with the blank group,the expression level of α-SMA in the model group was significantly decreased,and the protein expression levels of CRBP1,PCNA,MMP-9,TGF-β1,TβRI,p-Smad3 were significantly increased(P<0.05);compared with the model group,the serum containing HCXFZY decoction could increase the expression level of α-SMA in a concentration dependent manner,and inhibit the expression levels of CRBP-1,PCNA,MMP-9,TGF-β1,TβRI,p-Smad3(P<0.05);There was no significant difference in the total level of t-Smad3 among the groups(P>0.05).5.Immunofluorescence staining results:compared with the blank group,the model group significantly decreased the average immunofluorescence intensity of a-SMA and significantly increased the average immunofluoresce-nce intensity of CRBP1(P<0.05);compared with the model group,the HCXFZY decoction serum could enhance the average immunofluorescence intensity of α-SMA and weaken the average immunofluorescence intensity of CRBP1 in a concentration dependent manner(P<0.05).6.Through laser confocal localization observation,it was found that:compared with the blank group,the ratio of nucleus/cytoplasm of p-Smad3 in the model group was significantly increased;compared with the model group,with the increase of the concentration of HCXFZY decoction containing serum,the ratio of nucleus/cytoplasm of p-Smad3 was gradually decreased,and the nuclear translocation was reversed(P<0.05).7.The expression of PCNA mRNA and MMP-9 mRNA was detected by RT-PCR:compared with the blank group,the expression levels of PCNA mRNA and MMP-9 mRNA in the model group were significantly increased(P<0.05);compared with the model group,the expression levels of PCNA mRNA and MMP-9 mRNA were inhibited in a concentration dependent manner by HCXFZY decoction containing serum(P<0.05).Conclusion1.Using the platform of traditional Chinese medicine inheritance,we excavated professor Zhao Gang’s pathogenesis of lower extremity arterIoscler-osis obliterans.The treatment principle of activating blood circulation to remove blood stasis and paying equal attention to dredging and tonifying was put forward,and the basic core prescription of the disease was extracted,which was the decoction of cutting blood and removing stasis.2.HCXFZY decoction was used to treat ASO.After 4 weeks,the TCM symptom score,ABI index,infrared thermography,TG,TC,HDL-C,LDL-C,hs-CRP and FIB laboratory indexes were evaluated,and all outcome indexes were improved.And in the course of treatment,no obvious clinical adverse reactions.3.HCXFZY decoction can inhibit the phenotype transformation,proliferation and migration of VSMCs by regulating the expression of TGF-β1/Smad3 signaling pathway and related proteins,and delay the progression of ASO.