The Research On The Immune Pathogenesy And Regulation Of Myocardial Injury After Skeletal Muscle Ischemia Reperfusion

Background and Objective: The operation of extremities(such as total kneearthroplasty during which tourniquet is used) can cause skeletal muscle ischemiareperfusion injury which can trigger a systemic infammatory response syndrome(SIRS) and cause remote important organs dysfunction including heart, lung, liver andkidney. Clinical studies suggest that the heart is at very high risk in the setting ofSIRS. Our past research has demonstrated skeletal muscle ischemia reperfusion caninduce myocardial injury. The injury is the most serious in4-6hours afterreperfusion.The mechanism may be involved in the activating of neutrophil andinflammatory cytokines, excess oxidative stress, the imbalance of oxidant-antioxidantsystem, high secreting of aldosterone. However, the immune-regulating mechanismof myocardial injury after skeletal muscle ischemia reperfusion is not very clear.Toll-like receptors (TLRs) are the most important innate immune receptors and play apivotal role in initiating and shaping innate and adaptive immune responses. Recently,Toll-like receptor4(TLR4) have been shown to mediate the infammatory response inexperimental ischemia and reperfusion (I/R). TLR4involved in alerting the innateimmune system of danger seems to be activated by damage-associated molecularpattern molecules (DAMPs) that are released during IR. High-mobility group boxprotein1(HMGB1) is a very important DAMP. Myeloid differentiationprimary-response gene88(MyD88) is main adaptor protein of TLR4. TLR4andMyD88binding can activate downstream nuclear factor-kB (NF-kB) signaltransduction pathway and trigger inflammatory response. Role ofHMGB1-TLR4-MyD88-NF-kB signal pathway in myocardial injury after skeletalmuscle ischemia reperfusion is poorly understood.Our experiment is divided into two parts. Firstly, we observate myocardialinjury and the changes of innate and adaptive immune responses during total knee arthroplasty. Secondly, we have established the model of bilateral hindlimb ischemiaand reperfusion in rats by using a tourniquet and explored role ofHMGB1-TLR4-MyD88-NF-kB signal pathway and inflammatory response inmyocardial injury after skeletal muscle ischemia reperfusion. Moreover, we are tofurtherly ascertain the regulation roles of exogenous H2S donor NaHS andaldosterone receptor blocker spironolactone (spiron) on theHMGB1-TLR4-MyD88-NF-kB signal pathway.We also observateHMGB1-TLR4-MyD88-NF-kB signal pathway and inflammatory responses inmyocardial injury during rat skeletal muscle ischemia-reperfusion combined withfracture and soft tissue injury.Methods: In the first part, we selected15patients who were given double totalknee arthroplasty. Blood samples were drawn at the following time-points: beforeoperation, at4and12h after operation. Plasma troponin-I (TnI) and IL-6, IgG, IgM,IgA level in each group were measured. The flow cytometry technique was used todetect the changes of CD3+, CD4+, CD8+lymphocyte subsets and TLR4expressionof monocyte and neutrophil.Paired t-test was applied to statistics analysis. In thesecond part, forty-six Wistar rats were divided into6groups randomly:①Groupnormal (C),②Group IR:4h reperfusion following4h ischemia,③Group IR+trauma:IR+fracture and soft tissue injury,④Group IR+NaHS: IR+NaHS0.78mg/kg beforeischemia and reperfusion,ip,⑤Group IR+PPG: IR+DL-propargylglycine (PPG)60mg/kg before ischemia and reperfusion, ip,⑥Group IR+spiron. IR+Spiron20mg/kg/day for6days before ischemia,po. The apoptosis changes in themyocardium and plasma levels of TnI were observed. The values of plasma andmyocardial IL-6, IL-10, IL-17, HMGB1were measured. Western Blot method wasused to detect myocardial HMGB1, TLR4, MyD88, NF-κ Bp65, p-NF-κ Bp65relative protein content. Real-time PCR method was used to determine the expressionof rat myocardial HMGB1, TLR4, MyD88mRNA. Immunohistochemistry techniquewas used to determine expression of TLR4and p-NF-κ Bp65in myocardium.Results: The plasma TnI and IL-6levels were significantly increased in4h and12h post-operation compared with in pre-operation9(P<0.05). TLR4were mostlyexpressed in monocytes of peripheral blood, with relative low expression levels inneutrophils. The expression levels of TLR4in monocytes and neutrophils weresignificantly upregulated in4h and12h post-operation compared with in pre-operation(P<0.05).The levels of CD4+, CD4+/CD8+lymphocytes were markedlyincreased in12h post-operation compared with in pre-operation and in4hpost-operation(P<0.05). IgG, IgM, IgA levels were of no significance betweenpost-operation and pre-operation(P>0.05). Compared with Group normal, myocardialapoptotic index(AI), plasma TnI levels and the values of plasma and myocardial IL-6,IL-10, IL-17, HMGB1was significantly increased in Group IR(P<0.05). The relativeprotein content of HMGB1, TLR4, MyD88, p-NF-κ Bp65and myocardial HMGB1,TLR4, MyD88mRNA levels were significantly upregulated in group IR(P<0.05).Immunohistochemical results showed that the positive signals of TLR4and p-NF-κBp65was significantly increased in Group IR. Compared with Group IR, myocardialAI, plasma TnI levels and the values of plasma and myocardial IL-6, IL-17, HMGB1were obviously decreased in group IR+NaHS and group IR+spiron(P<0.05). Thevalues of plasma and myocardial IL-10were significantly increased. The relativeprotein content of HMGB1, TLR4, MyD88, p-NF-κ Bp65and myocardial HMGB1,TLR4, MyD88mRNA levels were significantly downregulated in group IR+NaHSand group IR+spiron(P<0.05). Immunohistochemical results showed that the positivesignals of TLR4and p-NF-κ Bp65was significantly decreased in IR+NaHS andgroup IR+spiron. Compared with Group IR, myocardial AI, plasma TnI levels and thevalues of plasma and myocardial IL-6, IL-17, HMGB1was significantly increased inGroup IR+PPG and Group IR+trama(P<0.05). The relative protein content ofHMGB1, TLR4, MyD88, p-NF-κ Bp65and myocardial HMGB1, TLR4, MyD88mRNA levels were significantly upregulated in group Group IR+PPG and GroupIR+trama(P<0.05). Immunohistochemical results showed that the positive signals ofTLR4and p-NF-κ Bp65was significantly increased in Group IR+PPG and GroupIR+trama. The relative protein content of NF-κ Bp65of all groups was of no significance(P>0.05).Conclusions:The operation of total knee arthroplasty can activate innateimmune and infammatory responses, inhibit cellular immunity and cause myocardialinjury.Skeletal muscle IR and IR combined trauma can activate myocardialHMGB1-TLR4-MyD88-NF-kB signal pathway，trigger systemic and myocardiallocal infammatory response and induce myocardial apoptosis and injury. ExogenousH2S donor NaHS and ALD receptor blocker spironolactone can alleviate myocardialapoptosis and injury though inhibiting myocardial HMGB1-TLR4-MyD88-NF-kBsignal pathway, downregulating proinfammatory cell factor IL-6,IL-17andupregulating anti-infammatory cell factor IL-10.It has been confirmed that thecombination of ALD and its receptor and the inhibiting of signal molecule H2S canactivate myocardial HMGB1-TLR4-MyD88-NF-kB innate immune signalpathway,mediate infammatory response and result in myocardial injury after skeletalmuscle ischemia reperfusion.