| ObjectiveSepsis can promote oxidative stress and inflammatory response to induce myocardial injury,reducing the level of inflammatory factors in cardiomyocytes through antioxidant/anti-inflammatory is an effective means to treat myocardial injury in sepsis.In this paper,we investigated the therapeutic effect and molecular mechanism of Shenfu Injection through retrospective clinical study,in vivo study(establishment of CLP sepsis myocardial injury rat model)and in vitro study(establishment of LPS-induced sepsis myocardial cell model)to provide scientific basis for the treatment of sepsis myocardial injury with Yi Qi Fu Zheng method.Methods1.Clinical study:patients with septic myocardial injury treated in the ICU from January 1,2020 to December 31,2022 in our hospital and the first affiliated hospital,with TCM evidence of heart-kidney Yang deficiency,a total of 76 patients who had used Shenfu injection in combination with basic treatment and those who applied basic treatment only were screened,and were divided into 38 cases in the observation group and 38 cases in the control group according to whether they were treated with Shenfu injection in combination.The myocardial injury markers and inflammatory indexes,critical illness score,TCM evidence of efficacy.2.In vivo study:84 SD rats were adaptively housed for 1 week,12 rats were randomly taken to give abdominal incision and suture as sham operation group,and the remaining 72 rats were given cecum ligation and puncture(CLP)to establish a model of myocardial injury in sepsis.After successful modeling,the rats were randomly divided into model group,NAC group(150 mg·kg-1),SF high-dose group(7.6 mL·kg-1),SF low-dose group(1.8mL·kg-1),SF high-dose+NAC group(7.6 mL·kg-1+150 mg·kg-1),SF low-dose+NAC group(1.8mL·kg-1+150 mg·kg-1),saline was given to the sham-operated group,and the corresponding doses were given to the remaining groups,administered continuously for 3 days.The survival rate of rats in each group was compared.Serum cTnI,CK-MB,BNP,IL-18,IL-1β,IL-10 levels were detected by Elisa assay,pathomorphological changes of cardiomyocytes were observed by HE staining,and the expression of oxidative factors and inflammatory factor-related proteins in cardiac tissues were detected by immunohistochemistry(IHC)and westernblotting(WB).3.In vitro study:LPS was used to stimulate human cardiomyocytes AC 16 to form a septic cardiomyocyte model.CCK8 method was used to detect the effect of different concentrations of LPS and Shenfu injection(SF)on the survival rate of AC16 at different times,and to screen the appropriate modeling and administration concentrations for subsequent experiments.According to the screening results,LPS(25 μg·mL-1)was used to stimulate AC 16 to form septic cardiomyocytes for 36 hours.The CCK8 method was used to detect the effect of the attached injection solution on the survival rate of LPS-induced AC 16.Fluorescent probe assay was used to detect the effect of sensual injection on LPS-induced intracellular ROS levels in AC 16s.The NLRP3 overexpression lentiviral vector and the control vector were transfected into AC 16 cells according to the lentiviral transfection procedure to obtain the NLRP3 overexpression group of AC 16 cell lines and the control group of AC 16 cell lines,and the WB method was used to detect whether the NLRP3 protein was successfully overexpressed in the NLRP3 overexpression group of AC 16 cell lines.and the experiment was divided into five groups:blank control group(referred to as blank group),NLRP3 vector control group(referred to as control group),SF+NLRP3 vector control group(referred to as SF group),NLRP3 overexpression group,and SF+NLRP3 overexpression group,except for the blank group,the remaining groups were treated with 25 μg·mL-1 LPS was used to stimulate the cells,and the cells were intervened with sensual injection for 36 h.Real-time quantitative PCR(RT-qPCR)and Westernblotting(WB)were used to detect the expression of mRNA and protein of NLRP3/inflammatory factor pathway-related genes.Results1.Clinical study:the results of myocardial markers after treatment in both groups showed that the levels of cTnI,NT-ProBNP and CK-MB decreased in the observation group compared with the control group,and the differences were statistically significant(P<0.01,P<0.05);the results of inflammatory indexes after treatment in both groups showed that the levels of CRP and IL-6 decreased in the observation group compared with the control group,and the differences were statistically significant(P<0.01,P<0.05);the results of APACHE Ⅱ score after treatment in both groups showed that compared with the control group,the APACHE Ⅱ score in the observation group decreased,and the difference was statistically significant(P<0.05);comparing the efficacy of Chinese medicine in both groups,the total effective rate in the observation group was 92.1%,and the total effective rate in the control group was 71.1%,and the difference was statistically significant(P<0.05).2.In vivo study:In the model of septic myocardial injury rats constructed,after 3 days of modeling administration,the survival rate of the sham-operated group was 100%,and the survival rate of the model group rats was 41.67%.while the survival rate of the remaining groups was improved,but the difference was not statistically significant when compared with the model group(P>0.05).Compared with the sham-operated group,the levels of cTnI,CK-MB,BNP,IL-18 and IL-1β were significantly higher and the level of IL-10 was significantly lower in the model group,and the difference was statistically significant(P<0.01).Compared with the model group,the use of Shenfu injection alone(high dose mainly)significantly reduced serum cTnI,CK-MB,BNP,IL-18,IL-1β levels,with statistically significant differences(P<0.01,P<0.05),and IL-10 levels were increased,but the differences were not statistically significant(P>0.05).The above-mentioned myocardial injury factors and inflammatory factors decreased more significantly after the combination of Shenfu injection of all dose and NAC,and the level of IL-10 could be increased,and the difference was statistically significant(P<0.01,P<0.05).Conventional HE staining showed that in the model group,the lesions were significantly increased,the myocardial tissue structure was obviously defective,the myofibrils were severely broken,and the lesions were surrounded by inflammatory exudation and hemorrhage,and NAC and Shenfu injection had different degrees of improvement at each dose.IHC showed that caspase-1 and IL-1β protein expressions were increased in the model group,and the difference was statistically significant(P<0.01),and all doses of Shenfu injection and combined NAC could reduce caspase-1 and IL-1βprotein expression levels,and the difference was statistically significant(P<0.01,P<0.05).pro-caspase-1,caspase-1,IL-1β,and IL-18 protein expression were upregulated in the model group,and the differences were statistically significant(P<0.01,P<0.05),and each dose of Shenfu injection and its combined NAC could significantly reduce the expression of NLRP3,pro-caspase-1,caspase-1,and IL-1 β protein,and Shenfu injection low dose and combined with NAC decreased the expression of NOX4,IL-18 protein,and the difference was statistically significant(P<0.01,P<0.05).3.In vitro study:CCK8 results showed no effect on cell survival when LPS stimulated at concentrations not higher than 25 μg·mL-1 for 24 hours compared with the blank group(P>0.05),and an extreme decrease in survival at concentrations above 50μg·mL-1,with statistically significant differences(P<0.01),which was not suitable for modeling.There was no effect on cell survival when the concentration of LPS stimulation for 36 hours was not higher than 10 μg·mL-1(P>0.05),and the survival rate decreased at concentrations above 25 μg·mL-1,with a statistically significant difference(P<0.01).The cell survival rate at 25 μg·mL-1 was between 70%and 80%,which was in accordance with the modeling conditions.Compared with the blank group,when the intervention concentration of Shenfu injection was above 20～40 μL·mL-1 on cell proliferation,the difference was statistically significant(P<0.01,P<0.05),with 40 μL·mL-1 having the highest cell survival rate,which could be used as the concentration for subsequent experiments.The cell survival rate of AC 16 was significantly increased after 36 hours of the intervention with LPS(25 μg·mL-1)and Shenfu injection(40 μL·mL-1),the difference was statistically significant(P<0.01).After transfecting AC 16 cells with NLRP3 overexpression lentiviral vector and control vector,the cells in the NLRP3 overexpression group showed significant overexpression of NLRP3 compared with the control group,and the difference was statistically significant(P<0.01),the AC16 cell line with stable overexpression of NLRP3 was successfully constructed.The results of the mechanism study showed that,compared with the blank group,the gene expression of NLRP3,ASC,caspase-1,and IL-1β was upregulated in the control and NLRP3 overexpression groups(P<0.01),while promoting the expression of NLRP3,ASC,caspase-1,IL-1β,and IL-18 proteins,with statistically significant differences(P<0.01,P<0.05).Compared with the control group,NLRP3,ASC,caspase-1,IL-1β gene expression was downregulated in the SF group(P<0.01,P<0.05),while NLRP3,ASC,caspase-1,IL-1β,IL-18 protein expression was inhibited,and the differences were statistically significant(P<0.01,P<0.05).Compared with the NLRP3 overexpression group,the gene expression of NLRP3,ASC,caspase-1,and IL-1 β was downregulated(P<0.01,P<0.05),while the expression of NLRP3,ASC,caspase-1,IL-1β,and IL-18 proteins was downregulated,with statistically significant differences(P<0.01，P<0.05).Compared with the SF group,the expression of NLRP3,ASC,caspase-1,and IL-1β was upregulated in the SF+NLRP3 control group(P<0.01,P<0.05),while the expression of NLRP3,ASC,caspase-1,and IL-18 proteins was promoted,with statistically significant differences(P<0.01,P<0.05),and the protein of IL-1β expression was also upregulated,but the difference was not statistically significant(P>0.05).ConclusionThe combination of Shenfu injection with conventional treatment can reduce myocardial damage,decrease the level of inflammatory factors,improve the systemic functional indexes and clinical symptoms.The generation of myocardial injury in sepsis may be related to excessive activation of NLRP3,and Shenfu injection may exert antioxidant and anti-inflammatory effects through inhibiting the NOX4/NLRP3/inflammatory factor pathway to reduce myocardial cell injury. |
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