Effect And Mechanism Of Nuanxinkang On Treatment Of Heart Failure Based On Mtln Regulation Of Macrophage Energy Metabolism Reprogramming

Objective:To investigate the role of Mtln-regulated macrophage energy metabolism reprogramming in heart failure and the intervention effect mediated by Nuanxinkang through Mtln.Methods:1.Animal experiments(1)Construction of Mtln Knockdown mice:Adult ROSA26-LSL-Cas9 Knockin mice were bred with LysM-Cre mice.Offspring were genetically identified and myeloid-bearing cells were screened for specific expression of LysM promoter-driven Cre recombinase Cas9 mice.Mtln-sgRNA was injected by tail vein injection.(2)A model of ischemia reperfusion(IR)injury-induced heart failure was established using reversible ligation of the left anterior descending coronary artery.(3)Animals were divided into four groups in the first part of the experiment:control group(Ctrl),Mtln knockdown group(Knockdown),control group+ischemia-reperfusion injury(Ctrl+ IR),and Mtln knockdown group+ischemia-reperfusion injury(Knockdown+IR).(4)Second part of the experiment①Transgenic mice were divided into six groups:control group(Ctrl),control group+IR injury(Ctrl+ IR),control group+IR injury+Nuanxinkang(Ctrl+IR+NXK),Mtln knockdown group(Knockdown),Mtln knockdown group+IR injury(Knockdown+IR),Mtln knockdown group+IR injury+Nuanxinkang(Knockdown+IR+NXK).Durg administration was performed on the second day after operation,and the dose of Nuanxinkang was 1.65g powder/kg/d.Ctrl+IR+NXK and knockdown+IR+NXK mice were gavaged continuously for 4 weeks using this dose.Ctrl,Ctrl+IR Knockdown,and Knockdown+IR were gavaged with equal amounts of saline.②C57BL/6 mice were divided into three groups:sham operation group(Sham),myocardial ischemia-reperfusion group(IR),and Nuanxinkang group(NXK).NXK administration dose was the same as before.2.Cell experiments(1)Cell culture:RAW264.7 cells were cultured in RPIM 1640 medium containing 10%FBS and 100 U/ml penicillin/100 mg/ml streptomycin,and 0.25%trypsin was digested and passaged when the cells were grown to 90%density in the culture dish.After cell passage,the culture medium was changed daily.(2)Cell transfection:Cells in the logarithmic growth phase were seeded in 6-well plates at about 2 × 105 cells per well and placed in an incubator for 12 h.The medium was changed and lentivirions(MOI of 20)and polybrene(5 μg/ml)were added;after 48 hours of infection,the cell culture medium used for infection was changed with new complete medium.When the cells grew to 90%density,they were transferred to 10-cm dishes.The transfected cells were divided into four groups.Mtln knockdown group(sh-Mtln)and corresponding control group(NC);Mtln overexpression group(OE-Mtln)and corresponding control group(LCK),Expression level of Mtln was examined after puromycin selection.(3)Extraction of bone marrow-derived macrophages:Bone marrow was isolated from the femurs and tibias of mice injected with AAV virus for 4 weeks to prepare single cells;the cells were seeded in culture flasks and placed in a cell incubator overnight;nonadherent cells were collected and re-seeded in new culture dishes(at this time,cells were cultured in RPMI 1640 medium containing 10%FBS and 1%100 U/ml penicillin/100 mg/ml streptomycin supplemented with 10 ng/mL of M-CSF);and cultured for 7 days to promote macrophage differentiation.The extracted BMDMs were divided into 2 groups:Ctrl and Knockdown group.(4)In the first part of the cell experiments,the groups are as follows according to the experimental needs:①RAW264.7 cells after successful transfection were stimulated with LPS(200 ng/mL)and IL-4(20 ng/mL)to detect changes in phenotype and energy metabolism.Grouping of RAW264.7 cells:NC,sh-Mtln,NC+IL-4,and sh-Mtln+IL-4.LCK,OEMtln,LCK+LPS,OE-Mtln+LPS.②Grouping of BMDMs:Ctrl,Knockdown,Ctrl+IL-4,Knockdown+IL-4.(5)Second part of the cell experimental grouping:①Grouping of RAW264.7 cells transfected with NC and sh-Mtln lentiviruses:NC(cultured with control serum),sh-Mtln(cultured with control serum),NC+LPS(cultured with control serum),sh-Mtln+LPS(cultured with control serum),NC+LPS+NXK(cultured with Nuanxinkang medicated serum),and sh-Mtln+LPS+NXK(cultured with Nuanxinkang medicated serum).②RAW264.7 cells transfected with LCK and OE-Mtln lentiviruses were treated as follows:LCK(cultured with control serum),OE-Mtln(cultured with control serum),LCK+LPS(cultured with control serum),OE-Mtln+LPS(cultured with control serum),LCK+LPS+NXK(cultured with Nuanxinkang medicated serum),and OE-Mtln+LPS+NXK(cultured with Nuanxinkang medicated serum).③Untransfected RAW264.7 was divided into three groups:control group(Con),LPS stimulation(LPS group),and given NXK-containing serum intervention after LPS stimulation(NXK group).3.Detection methods(1)Echocardiography was used to detect the parameters of cardiac function.(2)Hematoxylin-eosin(HE)staining were used to detect myocardial pathological structural changes.(3)Masson staining and Sirius red staining were used to observe myocardial fibrosis and collagen deposition.(4)Pro-inflammatory macrophages(M1)and anti-inflammatory macrophages(M2)changes in the hearts of mice after different treatments were detected by immunofluorescence.(5)qPCR was used to detect the mRNA expression levels of macrophage-associated proinflammatory cytokines Cxcl1,Cxcl2,TNF-α,IL-6,IL-1β and anti-inflammatory mediators FIZZ,MRC1,Argl,and IL-10 before and after modeling.(6)Flow cytometry was used to analyze the ratios of leukocytes,neutrophils,total macrophages,pro-inflammatory macrophages,and anti-inflammatory macrophages in unmodeled and modeled mice.(7)qPCR and/or Western blot were used to detect the expression levels of related inflammatory factors in transfected RAW264.7 cells and BMDMs under different conditions.(8)Seahorse to analyze the glycolytic,oxidative phosphorylation levels of transfected RAW264.7 cells,BMDMs under different conditions.(9)qPCR was used to detect the expression changes of energy metabolism-related enzymes SDHa,SDHb,SDHc,SDHd,PDH,OGDH,IDH1,IDH2,and IRG1 in infected RAW264.7 cells under different conditions.(10)The expression levels of IRG1,an enzyme related to energy metabolism,were detected by Western blot in infected RAW264.7 cells under different conditions.(11)The interaction between Mtln and IRG1 was detected by Co-Immunoprecipitation.Results:1.Results of the first part of the experiments(1)Before IR surgery,there was no significant difference in the levels of EF,FS,LVESV and LVEDV between Ctrl and Knockdown mice.At all stages after IR surgery,Mtln-KO+IR mice showed more prominent reduced EF and FS,and more significant enhanced LVESV and LVEDV levels compared with Ctrl+IR.(2)There was no difference in cardiac structure between Ctrl and Knockdown mice before IR.At 28 days after IR,the hearts of Knockdown+IR mice showed more significant myocardial structural destruction compared with Ctrl+ IR mice,including thinning of the myocardium,rupture,and dissolution of large areas of myocardial fibers,and a large increase in myocarditis cell infiltration.(3)Masson staining and Sirius staining were performed to determine the changes of fiber deposition.The results showed that no significant collagen fiber deposition was observed in the hearts of mice in the Ctrl and Knockdown group before IR.At 28 days after IR,cardiac collagen deposition was more pronounced in Knockdown+ IR mice compared with Ctrl+IR mice,and collagen fibers in the left ventricle replaced normal myocardium and formed a thinner wall structure.(4)Immunofluorescence staining was used to detect the changes of pro-inflammatory macrophages(M1)and anti-inflammatory macrophages(M2)at 28 days after IR.The results showed that M1 macrophages and M2 macrophages were not significantly infiltrated in the hearts of Ctrl and Knockdown mice before IR surgery.At 28 days after IR surgery,Knockdown+IR mice had increased cardiac M1 macrophages and decreased M2 macrophages compared with Ctrl+IR mice.(5)Before IR,there was no significant difference in the mRNA of pro-inflammatory related factors Cxcll,Cxcl2,TNF-α,IL-6,IL-1β and anti-inflammatory related factors FIZZ,MRC1,Arg1,and IL-10 in macrophages in the hearts of Ctrl and Knockdown mice.At 28 days after IR,Knockdown mice had significantly increased pro-inflammatory factor expression and significantly reduced anti-inflammatory factor expression compared with Ctrl mice.(6)Flow cytometric analysis showed no difference in the ratio of leukocytes,neutrophils,total macrophages,pro-inflammatory macrophages,and anti-inflammatory macrophages between Ctrl and Knockdown mice before IR.At 28 days after IR,the proportion of leukocytes,neutrophils,and total macrophages in the heart was not different in Knockdown mice compared with Ctrl mice,but the proportion of pro-inflammatory macrophages was significantly increased,and the ratio of anti-inflammatory macrophages was significantly reduced.(7)There was no difference in the expression of inflammatory cytokines INOS,IL-6,IL-1β,and TNF-α between LCK and OE-Mtln.Under LPS stimulation,compared with LCK.the expression of pro-inflammatory factors INOS,IL-6,IL-1β,and TNF-α was significantly decreased in RAW264.7 cells in the OE-Mtln group.Under IL-4 stimulation,the expression of MRC1.FIZZ,Arg1,and YM1 was significantly decreased in RAW264.7 cells in the sh-Mtln group compared with NC.(8)There was no significant difference in the mRNA expression levels of antiinflammatory cytokines FIZZ,MRC1,Arg1,YM1,and IL-10 in BMDMs between the Ctrl and sh-Mtln group.Upon IL-4 stimulation,the mRNA expression of FIZZ,MRC1,Argl,YM1,and IL-10 was significantly decreased in BMDMs of the sh-Mtln+IL-4 group compared with the Ctrl+IL-4 group.(9)In transfected RAW264.7 cells,upon IL-4 stimulation,the level of oxidative phosphorylation was significantly lower in the sh-Mtln group compared to NC.Consistent with this,even under LPS-stimulated conditions,the OE-Mtln group showed lower levels of oxidative phosphorylation compared to LCK.(10)The expression levels of SDHa,SDHb,SDHc,SDHd,PDH,OGDH,IDH1,IDH2,and IRG1 were detected by qPCR.The results showed that Mtln knockdown significantly inhibited SDHa,SDHb,SDHc,and SDHd expression levels and upregulated the expression level of IRG1 upon LPS stimulation.The results of Mtln overexpression were in contrast to this.(11)The protein expression level of IRG1 was detected by Western blot.The results showed that upon LPS stimulation,Mtln knockdown markedly upregulated the protein expression level of IRG1,and Mtln overexpression reversed this phenomenon.(12)The results of Co-Immunoprecipitation showed that there was an interaction between IRG1 and Mtln.2.Results of the second part of the experiments(1)Echocardiography showed that the application of NXK significantly improved cardiac,function after IR injury in Ctrl mice.When Mtln was knocked down,NXK had no significant effect on cardiac function in mice after IR injury.(2)HE staining showed that NXK significantly improved cardiac structural pathological changes after IR injury in Ctrl mice.When Mtln was knocked down,NXK had no significant effect on cardiac structure in mice after IR injury.(3)The results of Masson staining and Sirius staining showed that NXK significantly improved cardiac collagen deposition after IR injury in Ctrl mice.When Mtln was knocked down,NXK had no significant effect on collagen deposition in mouse hearts after IR injury.(4)qPCR results showed that NXK significantly reduced the mRNA levels of cardiac proinflammatory cytokines Cxcll,Cxcl2,TNF-α,IL-1β,and IL-6 after IR injury in Ctrl mice.When Mtln was knocked down,NXK had no significant effect on these inflammatory factors.(5)qPCR results showed that NXK inhibited the mRNA levels of cardiac factors FIZZ,MRC1,Arg1,and YM1 after IR injury in Ctrl mice.When Mtln was knocked down,NXK had no significant effect on these inflammatory mediators.(6)The results of flow cytometric analysis showed that in the acute inflammatory phase,NXK had an inhibitory effect on intracardiac proinflammatory macrophages after IR injury and a promoting effect on the level of anti-inflammatory macrophages.In the chronic inflammatory phase,NXK inhibited the levels of pro-inflammatory macrophages and anti-inflammatory macrophages in the heart after IR injury.When Mtln was knocked down,NXK had no significant effect on the changes of macrophage phenotype.(7)qPCR and Western blot results showed that NXK was able to reduce the level of proinflammatory factors in macrophages,and this effect was inhibited after Mtln knockdown.Consistent with this,no further reduced levels of inflammatory factors were shown upon Mtln overexpression in combination with NXK.(8)Seahorse energy metabolism analysis showed that NXK was able to reduce the glycolytic level of macrophages upon LPS stimulation,and this effect was inhibited after Mtln knockdown.Mtln overexpression in combination with NXK did not show further reduced glycolytic levels.(9)NXK inhibited the activation of NF-κB,a transcription factor downstream of macrophage polarization,through Mtln.Conclusion:1.Mtln could reprogram the energy metabolism process of macrophages,and then regulate macrophage-mediated inflammatory response,which plays an important role in the process of heart failure after IR injury.2.Nuanxinkang could up-regulate Mtln to modulate the immunometabolic process of macrophages,thus preventing heart failure.