| Objective:The research is to discuss the he mechanism of Yiqihuoxue drugs in the treatment of sepsis-induced myocardial dysfunction(SIMD)though the animal experiments,to explain that Yiqihuoxue drug play a therapeutic role though intestinal flora/Treg/NLRP3/inflammatory factors pathway.Methods:A rat model of SIMD was prepared by cecal ligation and puncture.It was randomly divided into SPF sham operation group(SPF-Sham),SPF sepsis rat model group(SPF-CLP),sham group,CLP group,and antibiotic group(Sham-k,CLP-k),Yiqihuoxue Chinese drug(Tongguan Capsule,TGC)group,TGC-k group.The 16S amplicon sequencing technique was used to observe the correlation between SIMD and intestinal flora imbalance,and the effects on intestinal flora in different environments.The level of Treg cells in peripheral blood was analyzed by flow cytometry(FCM).The cardiac function of septic rats was detected by ultrasound.The myocardial injury markers were detected by ELISA.Western Blot was used to detecte the expression of NLRP3,ASC,casepase-1.Results:1.The model of SIMD was prepared by CLP in SD rats.The survival time of septic rats was significantly increased after intragastric administration of Tongguan Capsule.The results of ultrasound showed that heart function of the rats in TGC-18h group and TGC-72h group were improved.Compared with the sham group rats,the level of myocardial injury markers(CK-MB,LDH,cTnT)was significantly decreased.The difference was statistically significant(P<0.05).Intestinal bacteria in the Sham group were consisted mainly of Bacteroidetes(61%),Firmicutes(29%),and Verrucomicrobia(9%).Intestinal bacteria in the CLP-18h group was composed of Bacteroidetes(14%),Firmieutes(48%),Proteobacteria(30%),and Verrucomicrobia(8%)?Intestinal bacteria in the CLP-72h group were composed of Bacteroidetes(13%),Firmi cutes(79%),Proteobacteria(7%),and Verrucomicrobia(1%).After the intervention of Tongguan Capsule,the main components of intestinal bacteria in TGC-18h group were Bacteroidetes(16%),Firmicutes(50%),Proteobacteria(26%),Verrucomicrobia(5%),and Actinobacteria(3%).The gut bacterial composition of the TGC-72h group was Bacteroidetes(17%),Firmicutes(66%),Proteobacteria(15%),Verrucomicrobia(1%),and Actinobacteria(1%).Flow cytometry results showed that the Treg cells in the TGC-18h group decreased compared with the CLP-18h group,and the difference was statistically significant(P<0.05).Compared with the CLP-72h group,the Treg cells in the TGC-72h group decreased,and the difference was statistically significant(P<0.05).At the same time,the expression levels of NLRP-3 pathway-related proteins NLRP-3,ASC and Casepase-1 in the myocardial tissues of TGC-18h group and TGC-72h group decreased,the difference was statistically significant(P<0.05).The expression level of proteins NLRP-3,ASC and Casepase-1 were gradually decreased and the difference was statistically significant(P<0.05).The levels of serum inflammatory factors in TGC groups(IL?6?IL-1??IL-10?IL-18?TGF-?1)were significantly decreased.The difference was statistically significant(P<0.05).2.Antibiotics were intervened in rats to observe the role of gut bacteria in SIMD.The mortality of septic rats intervened by antibiotics was extremely high,compared with the rats in the Sham-K group.The difference was statistically significant(P<0.05).However,the mortality of septic rats increased after intragastric administration of Tongguan Capsules(TGC),and the difference was statistically significant(P<0.05).Ultrasound results showed that the cardiac function of rats with sepsis was significantly decreased,the levels of myocardial injury markers(CK-MB,LDH,cTnT)were significantly increased,and the difference was statistically significant(P<0.05).The intestinal flora of rats after antibiotic intervention had cleared mostly.The gut bacterial composition of the Sham-K group was composed of Proteobacteria(93%),and Tenericutes(7%).The CLP-18h-K was composed of Proteobacteria(100%).The CLP-72h-K was composed of Proteobacteria(70%),and Firmicutes(30%).After the administration of TGC,the cardiac function of rats with sepsis increased,the levels of myocardial injury markers(CK-MB,LDH,eTnT)decreased significantly,and the difference was statistically significant(P<0.05).The results of intestinal flora showed that under the intervention of Tongguan Capsule,the composition of intestinal bacteria in TGC-18h-K group was still Proteobacteria,accounting for 100%.The intestinal bacterial composition of the TGC-72h-K group was still Proteobacteria,accounting for 70%,and Firmicutes accounted for 30%.Flow cytometry results showed that the level of Treg cells in the CLP-18h-K group decreased significantly compared with the Sham-K group,the difference was statistically significant(P<0.05),with the prolongation of modeling time,CLP-72h-K group The level of Treg cells increased significantly,and the difference was statistically significant(P<0.05).Compared with the CLP-18h-K group,the level of Treg cells in the CLP-72 h-K group was increased,and the difference was statistically significant(P<0.05).After the administration of TGC,the Treg cells in the TGC-18h-K group increased significantly compared with the CLP-18h-K group after intervention with Tongguan Capsule(P<0.05).Compared with the CLP-72h-K group,the TGC cells in the TGC-72h-K group increased,and the difference was not statistically significant.The Treg cells in the TGC-72h-K group were significantly higher than those in the TGC-18h-K group(P<0.05).Meanwhile,the expression levels of NLRP-3 pathway-related proteins NLRP-3,ASC and Casepase-1 in myocardial tissue of septic rats increased,and the expression level increased gradually with the prolongation of modeling time.The difference was statistically significant(P<0.05).).Compared with the sham group rats,the expression levels of NLRP-3,ASC and Casepase-1 increased,and the difference was statistically significant(P<0.05).After the administration of TGC,the expression levels of NLRP-3,ASC and Casepase-1 in the myocardial tissue of TGC-18h-K group and TGC-72h-K group decreased,the difference was statistically significant(P<0.05),and The expression time was gradually decreased and the difference was statistically significant(P<0.05).The level of serum inflammatory factors(IL-6?IL-1??IL-10?IL-18?TGF-?1)was significantly decreased,and the difference was statistically significant(P<0.05).3.Rats were placed in SPF and normal grades,and CLP was performed at the same time.The performances of SMID in two class rats were similar.But the changes of intestinal flora were significant.The intestinal flora of the SPF-Sham group was mainly composed of Bacteroidetes(58%),Firmicutes(28%),and Verrucomicrobia(13%).The SPF-CLP-18h group was mainly composed of Bacteroidetes(17%),Firmicutes(42%)?Proteobacteria(34%),and Verrucomicrobia(7%).Intestinal bacteria in the SPF-CLP-72h group were composed of Bacteroidetes(38%),Firmicutes(54%)?Proteobacteria(6%),and Verrucomicrobia(2%).Flow cytometry results showed that the Treg cells in the SPF-CLP-18h group and the SPF-CLP-72h group increased compared with the SPF-Sham group,and the difference was statistically significant(P<0.05).Compared with the SPF-CLP-18h group,the Treg cells in the SPF-CLP-72h group increased,and the difference was statistically significant(P<0.05).At the same time,the expression levels of NLRP-3 pathway-related proteins NLRP-3,ASC and Casepase-1 in the myocardial tissues in SPF-CLP-18h group and SPF-CLP-72h group were increasing.The difference was statistically significant(P<0.05).Compared with the SPF-CLP-18h group,the expression level of proteins NLRP-3,ASC and Casepase-1 were gradually decreased in SPF-CLP-72h group,and the difference was statistically significant(P<0.05).The levels of serum pro-inflammatory factors(IL-6,IL-1?,IL-18)were significantly increased,and decreased in SPF-CLP-72h group,and the difference was statistically significant(P<0.05).Conelusion:1.In the rat model of SIMD by CLP,the performance of sepsis was accords with the study.The expression level of myocardial injury markers in serum was significantly increased.Ultrasound results show diastolic dysfunction and systolic dysfunction in rats,consistented with the performance of SIMD in the paper.2.Tongguan Capsule can significantly improve the cardiac function of rats with SIMD.The experimental results show that the therapeutic effect was to regulate the level of Treg cells and its anti-inflammatory factors and the expression of NLRP3 inflamnasome by regulating the balance of intestinal flora.3.Broad-spectrum antibiotics were used to eliminate most of the intestinal flora.And then the rat model of SIMD was made by CLP.Tongguan Capsule was intragastric administration.The experimental results showed that TGC can be manifested in prolonged survival times,improved the diastolic function and systolic function.As well as Treg cell levels and their anti-inflammatory factor levels and the expression of NLRP3 inflammasome by regulating the balance of intestinal flora.The most important thing was that Tongguan Capsule intervention could significantly improve the structure and proportion of intestinal flora,suggesting that Tongguan Capsule regulated Treg cell level and its anti-inflammatory factor level and the expression of NLRP3 inflammasome by regulating the balance of intestinal flora.4.After 16s test,it was found that the feeding conditions in different environments affected the intestinal flora of rats.CLP surgery maybe was the main reason for the changes in the structure and richness of intestinal flora in rats with sepsis cardiomyopathy.5.In the rat model of SIMD by CLP,the intestinal flora was significantly changed.Broad-spectrum antibiotics were used to eliminate most of the survival time was significantly reduced.Therefore,the intestinal flora is associated with SIMD,which is consistent with the "heart-spleen correlation theory”. |
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