Study On The Mechanism Of Huoxue Wentong Formula Combined With BMSCs Transplantation To Improve The Ischemic And Hypoxic Microenvironment After Myocardial Infarction

Background:The rapid development of contemporary regenerative medicine with stem cell technology as the core has brought new hopes for the treatment of myocardial infarction.Stem cell transplantation for the treatment of myocardial infarction is expected to repair necrotic myocardial tissue and improve the damaged heart function.The local microenvironment of ischemia and hypoxia in the infarct area reduces the survival rate of MSCs after transplantation,which greatly limits the efficacy of MSCs in the treatment of MI.The study of traditional Chinese medicine in promoting the survival of MSCs after transplantation has also shown a good effect.It can regulate the homing and colonization of MSCs and improve the survival rate after MSCs transplantation.Professor Yao Kuiwu,the tutor,based on the basic pathogenesis of "Yang Wei Yin Xian" of chest stuffiness and pains in TCM,often treated coronary heart disease by activating blood and warming heart-yang therapy,which had significant clinical effect.Based on many years of clinical experience,he formulated an empirical prescription with the effect of activating blood and warming heart-yang—Huoxue Wentong Formula.Our previous research also found that Huoxue Wentong Formula can significantly improve the heart function of myocardial ischemia model rats,significantly reduce the area of myocardial infarction,and can reduce inflammation,inhibit myocardial cell apoptosis,reduce the area of collagen fibers,and promote angiogenesis.The reduction of myocardial.ischemia injury by Huoxue Wentong Formula may be related to the activation of Sirtl signaling pathway.There is no relevant report on the treatment of myocardial infarction with Huoxue Wentong Formula combined with MSCs transplantation.Objective:1.To sum up the medication rules and treatment ideas of Professor Yao Kuiwu's to treat Yang-deficiency syndrome of coronary heart disease by activating blood and warming heart-yang therapy,and systematically summarizing the experience.2.To explore the effect of Huoxue Wentong Formula on BMSCs transplantation in the treatment of myocardial infarction and its possible mechanismMethods:1.From January 2016 to December 2020,we collected all the original medical records of the first diagnoses that were treated by the chief physician Yao Kuiwu,Guang'anmen Hospital,China Academy of Chinese Medical Sciences,treated with Chinese medicine decoctions and met the criteria for inclusion and discharge.Data analysis is carried out through the "Traditional Chinese Medicine Inheritance Computing Platform(V3.0)"software system.According to the analysis,the frequency of traditional Chinese medicine,commonly used combinations of traditional Chinese medicine,potential prescriptions,etc.,were obtained.The medication rules,diagnosis and treatment ideas of Professor Yao Kuiwu's therapy of activating blood and warming heart-yang in the treatment of coronary heart disease were analyzed and discussed,and a systematic summary was made.Based on the analysis of the frequency of Chinese herbal medicine,commonly used the traditional Chinese medicine composition,potential prescriptions,etc.,analyze and explore the medication rules and diagnosis and treatment ideas of Professor Yao Kuiwu's activating blood and warming heart-yang therapy for the treatment of coronary heart disease,and make a systematic summary.2.BMSCs were extracted from rats and cultured to the third generation,and the cells were identified by Reumann Cytometer.Cell survival after transplantation was detected by fluorescence labeling with living cell stain CM-DIL.3.Myocardial infarction model was established by ligation of the anterior descending branch of the left coronary artery.SD rats were randomly divided into 5 groups:Sham group:coronary artery was only threaded without ligation,and pure water was given by gavage after surgery for 28 days.Myocardial infarction group(MI group):model was made according to AMI modeling method,and 100uL PBS was directly injected into 2 sites of myocardial tissue at the edge of the infarction area after successful modeling.Pure water was gavaged for 28 days after operation.BMSCs transplantation group(BMSCs group):The model was made according to the AMI modeling method.After the model was successfully made,a total of 100ul MSCs single cell suspension(cell concentration 3x106 cells/100ul)was directly injected into 2 sites of myocardial tissue at the edge of the infarction area.Pure water was given intragastrically for 28 days.Huoxue Wentong formula group(HXWTF group):The modeling method was the same as that of myocardial infarction group.Postoperative gavage of Huoxue Wentong formula for 28 consecutive days;Huoxue Wentong Formula+BMSCs transplantation group(BMSCs+HXWTF group):The modeling method was the same as that of BMSCs transplantation group.Postoperative gavage of Huoxue Wentong formula for 28 consecutive days.4.Cardiac function of rats was detected by echocardiography.TTC staining was used to observe the size of myocardial infarction,and HE staining was used to observe the changes of myocardial tissue morphology in rats with myocardial infarction.The survival of BMSCs after transplantation was observed under fluorescence microscope.5.Serum SOD activity of rats was determined by WST-1 method and serum MDA level was determined by TBA method to evaluate the degree of oxidative damage in each group.Cardiomyocyte apoptosis was observed by TUNEL staining.The serum levels of inflammatory factors TNF-?,IL-6,IL-10 and growth factors VEGF and bFGF were determined by ELISA kit.The degree of myocardial fibrosis was detected by Sirius scarlet staining.The expression of ?-SMA and CD31 at the edge of infarction was detected by immunohi stochemi c al staining to evaluate angiogenesis.The protein expression levels of SIRT1,NF-KB,FoxO1 and p53 in myocardial tissue were detected by Western blotting.Results:1.From the analysis of traditional Chinese medicine frequency,among 196 prescriptions,there were a total of 137 TCM medicines,39 TCM medicines with medication frequency>20,and 22 TCM medicines with medication frequency?50.TheTCM medicines with medication frequency?50 from high to low were Salvia miltiorrhiza(193),Cinmanthus cassia(164),Amosum sanguinus(123),Ligusticum chuanxiong(117),Trichosanthis trichosanthis(109),Paeonia lactiflora(105),Main licorice root(95),caulis spatholobi(93),poria cocos(88),radix bupleuri(87),Fried hovenia dulcis(80),Chinese angelica(78),dried ginger(77),Fried atractylodes(76),eucommia bark(64),the codonopsis pilosula(61),the dwarf lilyturf(59),c.deserticola(58),Fried zizyphus jujube kernel(56),scallions white(54),puerarin(54),fruit of Chinese magnoliavine(50).2.The 137 traditional Chinese medicines in the 196 prescriptions are divided into 14 categories according to their efficacy.The top 8 in descending order according to the efficacy of the drugs are tonic(592),activating blood and removing blood stasis(461),and Wenli.(429),phlegm(158),dampness(151),Qi regulation(130),diuresis and dampness(120),rheumatism(81).2.The 137 traditional Chinese medicines in 196 prescriptions were divided into 16 categories according to their efficacy.The top 10 medicines in order of use frequency of drug efficacy were:tonifying deficiency(769),promoting blood circulation and removing blood stasis(436),warming Li(339),clearing heat(210),dispelling dampness(198),relieving surface(184),regulating qi(177),and so on.Phlegm-reducing class(155),water-permeating class(119),tranquilizing class(114).3.From the analysis of TCM combination frequency,the descending order of TCM combination frequency?90 was Salvia miltiorrhiza-Cinnamomum rhizome(162),Salvia miltiorrhiza-Amorum rhizome(123),Salvia miltiorrhiza-Ligusticum chuanxiong(115),Salvia miltiorrhiza-Trichosanthis(108),Salvia miltiorrhiza-Paeonia lactiflora(104),Salvia miltiorrhiza-Cinnamomum rhizome(99).Cassia twig-Amomum(99).Based on association rules,the traditional Chinese medicine combinations were deeply analyzed.The commonly used two traditional Chinese medicine combinations in the treatment of coronary heart disease by the therapy of activating blood and warming heart-yang were Salvia miltiorrhiza-Trichosanthis,Salvia miltiorrhiza-Amorum kernel,Salvia miltiorrhiza-Cinmae branch,Salvia miltiorrhiza-Coriolis spatholobi,etc.The combination of 3 drugs is Salvia miltiorrhiza-Amorum kernel-Trichosanthis,Salvia miltiorrhiza-Cinnamon twigs-Trichosanthis,Salvia miltiorrhiza-Cinnamon twigs-Cucumis spatholobus and so on.From the network topology of traditional Chinese medicine association rules,it can be seen that the traditional Chinese medicine combinations with relatively high correlation were Salvia miltiorrhiza,cassia twood,caulis spatholobi,Trichosanthis trichosanthis,Ligusticum chuanxiong and Amodium aurum.4.Cluster analysis based on complex algorithm can find core combinations commonly used in the treatment of coronary heart disease by tutoring the therapy of activating blood and warming heart-yang.The core clusters of three traditional Chinese medicines were Salvia miltiorrhiza,Cinmanthus cassia,Amothecum sanguinus,Trichosanthis and Caulis spatholobi.Salvia miltiorrhiza,cassia twiggy,Ligusticum chuanxiong,Paeonia paeoniae,Amomum kernel;Salvia miltiorrhiza,cassia twiggy,Ligusticum chuanxiong,Amosum kernels,Poria cocos.The core combinations of four traditional Chinese medicines were:Salvia miltiorrhiza,cassia twigs,Amorum seed,Trichosanthis trichosanthis,Ligusticum chuanxiong,Caulis spatholobi;Salvia miltiorrhiza,cassia twiggy,Radix Ophiopogonis,prepared liquorice,Ligusticum chuanxiong,rehmannia glutinosa;Salvia miltiorrhiza,cassia twood,asparagus,caulis spatholobi,Ligusticum chuanxiong,incense;Salvia miltiorrhiza,cassia twigs,Amosum kernels,Dried Ginger,Poria cocos,Trichosanthis.The core combinations of 5 traditional Chinese medicines were as follows.Salvia miltiorrhiza,cassia twigs,caulis spatholobi,Trichosanthes trichosanthes,Ligusticum chuanxiong,Dangshen;Cassia twiggy,Salvia miltiorrhiza,Radix Ophiopogonis,Ligusticum chuanxiong,Glycyrrhiza,Atractylodes Atractylodes;Salvia miltiorrhiza,cassia twood,dried ginger,Amomum kernels,Poria cocos,Coptis coptidis;Salvia miltiorrhiza,cassia twigs,Amorum kernels,Trichosanthis trichosanthis,Ligusticum chuanxiong,Paeonia lactiflora;Salvia miltiorrhiza,Amorum kernel,Cinnamon twiggy,Ligusticum chuanxiong,Caulis spatholobi,Dendrosia.5.Based on the analysis of prescription frequency,a total of 54 prescriptions were involved in 196 cases,including 32 prescriptions with a prescription frequency of more than 2,24 prescriptions with a prescription frequency of more than 5,13 prescriptions with a prescription frequency of more than 10,and 6 prescriptions with a prescription frequency of more than 20.It can be seen that the commonly used the therapy of activating blood and warming heart-yang for the treatment of coronary heart disease include Danshen Yin(118),Sini Powder(73),Guizhi Glycyrhiza Longgu Oyster Decoction(25),Linggui Zhugan Decoction(23),Guilou Xiebai Baijiu Decoction(21),etc.6.According to the use frequency of prescription compatibility,it can be concluded that the commonly used prescription compatibility for the treatment of coronary heart disease by the therapy of activating blood and warming heart-yang by the director is Danshen drink-Sini powder(47),Danshen drink-Huangqi Chifeng soup(11),Danshen drink-Jiaotai pill(11),Danshen drink-Guilou Xiebai Baijiu soup(11),Danshen drink-Banxia Xiexin soup(11),Salvia miltiorrhiza-Guilou Xiebai Pinellia Decoction(11),etc.Based on association rules,in-depth analysis of prescription combinations showed that the commonly used prescriptions for the treatment of coronary heart disease by the therapy of activating blood and warming heart-yang were Danshen Yin-Sini San-Guilou Xiebai Pinelaria Decoction,Danshen Yin-Sini San-Huangqi Chifeng Decoction,Danshen Yin-Sini San-Guizhi Glycyrhizoma Longgu Oysteria Decoction,etc.According to the network topology diagram of association rules of prescription combination rules,it can be seen that the prescriptions with relatively large correlation are Danshen Yin,Sini San,Shengmai San,Guizhi Glycyrhiza Longgu Oyster Decoction,Guilou Xiebai Pinellia Decoction,Guilou Xiebai Baijiu Decoction,Jiaotai pill,etc.Cluster analysis based on complex algorithm in the in-depth mining can be concluded that the core prescription combination commonly used in the treatment of coronary heart disease by the therapy of activating blood and warming heart-yang is Shengmai San,Danshen Yin,Zhishi Xiebai Guizhi Decoction,Buzhong Yiqi Decoction,Sijunzi Decoction;Salvia miltiorrhiza drink,Jiaotai pill,Pinellia Xiexin soup,Linggui Shu Gan soup,Yue Ju pill;Salvia miltiorrhiza drink,sini powder,astragalus red wind soup,Guilou stemon white Pinelaria soup,cassia twig-licorice dragon bone oyster soup.7.Identification of BMSCs:The expression of CD44,CD90 and CD45 surface markers of the third generation of BMSCs was detected by Reutersi cytometer.The results showed that the expression rates of CD44,CD90 and CD45 were 99.2%,97.4%and 6.7%respectively.8.Effects of Huoxue Wentong Formula combined with MSCs transplantation on cardiac function in rats with myocardial infarction:Compared with MI group,BMSCs transplantation group,Huoxue Wentong Formula group,and Huoxue Wentong Formula combined with BMSCs transplantation group had significant improvement in EF(**P<0.01,***P<0.001,***P<0.001).EF of Huoxue Wentong Formula combined with BMSCs transplantation group was significantly improved than that of BMSCs transplantation group and Huoxue Wentong Formula group(***P<0.001,**P<0.01);Compared with MI group,FS was significantly improved in BMSCs transplantation group,Huoxue Wentong Formula group,and Huoxue Wentong Formula combined with BMSCs transplantation group(**P<0.01,***P<0.001,***P<0.001).FS of Huoxue Wentong Formula combined with BMSCs transplantation group was significantly improved than that of BMSCs transplantation group and Huoxue Wentong Formula group(***P<0.001,***P<0.001).9.Effect of Huoxue Wentong Formula combined with MSCs transplantation on myocardial infarction area in rats with myocardial infarction:there was no infarction in the sham group,and the complete heart surface and heart sections were red without pale areas.In the MI group,both the surface of the intact heart and the pale area of the heart section were the largest,so the area of the MI was the largest,and the MI was obviously concave,and the myocardium at the MI was obviously thinned.Compared with MI group,BMSCs transplantation group and Huoxue Wentong Formula group showed significant reduction in the surface of intact heart and the pale area of heart sections,indicating that BMSCs transplantation and Huoxue Wentong Formula drug intervention can both reduce MI area.The huoxue wentong formula combined with BMSCs transplantation group had the smallest pale area in the heart and slight indentation in the myocardial infarction,indicating that huoxue wentong formula could improve the efficacy of BMSCs transplantation in improving the myocardial infarction area in rats with myocardial infarction.10.Effect of Huoxue Wentong Formula combined with MSCs transplantation on the morphology of myocardial tissue in rats with myocardial infarction:Compared with the sham operation group,HE staining showed that there were different degrees of inflammatory cell infiltration in the infarct area in the other groups.Myocardial infarction group cell arrangement disorder,cell space increased,inflammatory cell infiltration is the most serious.BMSCs transplantation group,Huoxue Wentong formula group,and Huoxue Wentong formula combined with BMSCs transplantation group showed significant improvement in these lesions,and inflammatory cell infiltration was reduced to varying degrees.Compared with the BMSCs transplantation group and the Huoxue Wentong Formula group,the Huoxue Wentong Formula combined with BMSCs transplantation group had more orderly cell arrangement and less inflammatory cell infiltration.11.Effect of Huoxue Wentong Formula on survival after BMSCs transplantation for four weeks:observed under a panoramic digital scanning microscope,it can be seen that the red fluorescence distribution of the Huoxue Wentong Formula combined with BMSCs transplantation group was wider.The amount of overlap between red fluorescence and blue fluorescence was significantly more.12.Effects of Huoxue Wentong Formula combined with BMSCs transplantation on SOD and MDA in serum of myocardial infarction rats:Compared with MI group,SOD activity in BMSCs transplantation group,Huoxue Wentong Formula group and Huoxue Wentong Formula combined with BMSCs transplantation group were significantly increased(**P<0.01,***P<0.001,***P<0.001);Compared with BMSCs transplantation group and Huoxue Wentong formula group,SOD activity in Huoxue Wentong formula combined with BMSCs transplantation group was significantly increased(***P<0.001,**P<0.01);Compared with MI group,MDA levels in BMSCs transplantation group,Huoxue Wentong Formula group,and Huoxue Wentong Formula combined with BMSCs transplantation group were significantly decreased(**P<0.01,***P<0.001,***P<0.001);Compared with the BMSCs transplantation group and the Huoxue Wentong Formula group,the MDA in the Huoxue Wentong Formula combined with BMSCs transplantation group was significantly decreased(***P<0.001,**P<0.01).13.Effect of Huoxue Wentong Formula combined with BMSCs transplantation on myocardial apoptosis in rats with myocardial infarction:Compared with MI group,the apoptosis level of BMSCs transplantation group,Huoxue Wentong Formula group and Huoxue Wentong Formula combined with BMSCs transplantation group was significantly decreased(***P<0.001,***P<0.001,***P<0.001);Compared with the BMSCs transplantation group and the Huoxue Wentong Formula group,the apoptosis level in the Huoxue Wentong Formula combined with BMSCs transplantation group was significantly decreased(**P<0.001,***P<0.001).14.Effects of Huoxue Wentong Formula combined with BMSCs transplantation on serum inflammatory factors TNF-?,IL-6 and IL-10 in myocardial infarction rats:Compared with the sham operation group,the levels of TNF-?,IL-6 and IL-10 in MI group were significantly increased(***P<0.001);Compared with M.I group,TNF-? was significantly decreased in BMSCs transplantation group,Huoxue Wentong Formula group,Huoxue Wentong Formula combined with BMSCs transplantation group(**P<0.01,***P<0.01,***P<0.001),and IL-6 was significantly decreased(***P<0.001,***P<0.001,***P<0.001),IL-10 significantly increased(***P<0.001,***P<0.001,***P<0.001);Compared with BMSCs transplantation group and Huoxue Wentong formula group,TNF-?,IL-6 and IL-10 in Huoxue Wentong formula combined with BMSCs transplantation group were significantly decreased(**P<0.01,**P<0.01),significantly decreased(***P<0.001,**P<0.01),and significantly increased(***P<0.001).***P<0.001).15.Effect of Huoxue Wentong Formula combined with BMSCs transplantation on myocardial fibrosis in rats with myocardial infarction:Compared with the sham operation group,myocardial fibrosis was obvious in the MI group(***P<0.001);Compared with the MI group,the area of myocardial fibrosis was significantly decreased in the BMSCs transplantation group,the Huoxue Wentong Formula group,and the Huoxue Wentong Formula combined with BMSCs transplantation group(***P<0.001,***P<0.001,***P<0.001);Compared with BMSCs transplantation group and Huoxue Wentong Formula group,the area of myocardial fibrosis in Huoxue Wentong Formula combined with BMSCs transplantation group was significantly reduced(*P<0.05,**P<0.01).16.The effect of Huoxue Wentong formula combined with BMSCs transplantation on the expression of ?-SMA and CD3 1 in MI rats:Compared with the sham operation group,the expression of ?-SMA and CD31 in MI group was significantly increased(***P<0.001,**P<0.01);Compared with the MI group,the expressions of ?-SMA and CD31 in the BMSCs transplantation group,the Huoxue Wentong Formula group and the Huoxue Wentong Formula combined with BMSCs transplantation group were significantly increased(***P<0.001,***P<0.001,***P<0.001).Compared with BMSCs transplantation group and Huoxue Wentong formula group,the expressions of ?-SMA and CD31 in Huoxue Wentong formula combined with BMSCs transplantation group were significantly increased(**P<0.01,*P<0.05;*P<0.05,***P<0.001).17.Effects of Huoxue Wentong Formula combined with BMSCs transplantation on serum growth factors VEGF and bFGF in myocardial infarction rats:Compared with the sham operation group,the levels of VEGF and bFGF in MI group were significantly increased(***P<0.001);Compared with MI group,VEGF and bFGF in BMSCs transplantation group,Huoxue Wentong Formula group,and Huoxue Wentong Formula combined with BMSCs transplantation group were significantly increased(***P<0.001,***P<0.001,***P<0.001);Compared with BMSCs transplantation group and Huoxue Wentong Formula group,VEGF and bFGF in Huoxue Wentong Formula combined with BMSCs transplantation group were significantly increased(***P<0.001,***P<0.001).18.Effects of Huoxue Wentong Formula combined with BMSCs transplantation on the protein expressions of SIRT1,NF-kB,FoxO1 and p53 in myocardial tissue:Compared with the sham operation group,the expression of SIRT1 was significantly decreased in the MI group(***P<0.001),and the expressions of NF-kB,FoxO1 and p53 were significantly increased(**P<0.01,**P<0.01,**P<0.01).Compared with MI group,the expression of SIRT1 in BMSCs transplantation group,Huoxue Wentong Formula group,and Huoxue Wentong Formula combined with BMSCs transplantation group was significantly increased(**P<0.01,***P<0.001,***P<0.00 1),and the expression of NF-?B was significantly decreased(*P<0.05,**P<0.01,**P<0.01),the expression of FoxO1 was significantly decreased(*P<0.05,*P<0.05,*P<0.05),and the expression of p53 was significantly decreased(*P<0.05,**P<0.01,**P<0.01);Compared with BMSCs transplantation group and Huoxue Wentong formula group,the expression of SIRT1 in Huoxue Wentong formula combined with BMSCs transplantation group was significantly increased(**P<0.01,*P<0.05),the expression of NF-kB was significantly decreased(**P<0.01,*P<0.05),and the expression of FoxO1 was significantly decreased(*P<0.05,*P<0.05),the expression of p53 was significantly decreased(*P<0.05,*P<0.05).Conclusion:1.The teacher's therapy of activating blood and warming heart-yang for the treatment of coronary heart disease pays equal attention to both constant blood circulation medicine and warming Yang medicine,and the use of traditional Chinese medicine is gentle,compatibility is clear,mainly by the prescription.Based on the traditional Chinese medicine inheritance calculation platform,three core drug combinations of the therapy of activating blood and warming heart-yang for the treatment of coronary heart disease were obtained through data mining,which were all similar to the drug composition of Huoxue Wentong formula.The clinical treatment effect of the instructor's therapy of activating blood and warming heart-yang is very effective in a short term.In the short term,it may have little effect on the large vessels,which may be due to the improvement of the myocardial ischemia and hypoxia microenvironment and the promotion of microangiogenesis.It also preliminarily shows that the clinical effect of Huoxue Wentong formula is quite remarkable2.Huoxue Wentong Formula may improve the survival rate after BMSCs transplantation by improving the ischemic hypoxic microenvironment after myocardial infarction,and further improve the efficacy of BMSCs transplantation in the treatment of myocardial infarction in terms of anti-cell oxidative damage,anti-cell apoptosis,reducing inflammatory response,anti-myocardial fibrosis and promoting angiogenesis,And it also promotes paracrine function of BMSCs after transplantation,further repairs damaged myocardium,reduces myocardial infarction area,and improves cardiac function.3.Huoxue Wentong formula can promote the activation of SIRT1 signaling pathway after BMSCs transplantation to synergically restore cardiac function.