| The China Cardiovascular Disease Report 2020 shows that the prevalence and mortality rate of cardiovascular disease in China is rising,and has become the number one killer that takes the lives of the nation,according to incomplete statistics,there are 330 million patients with cardiovascular disease in China,including 11.85 million patients with coronary heart disease.2 Cardiovascular disease is a great burden on our country and the world economy,and has become a major social public health problem.Myocardial ischemia is an important pathological manifestation of cardiovascular disease,which can cause irreversible myocardial damage and eventually evolve into myocardial infarction.The most effective clinical treatment for myocardial infarction is timely reperfusion to salvage the ischemic myocardium,including percutaneous coronary intervention(PCI)and pharmacological thrombolytic therapy.Real-world studies have shown that reperfusion therapy is a double-edged sword,and myocardial injury can be exacerbated by the resupply of blood flow to the ischemic myocardium after reperfusion,and the myocardial tissue damage caused by such reperfusion therapy is called myocardial ischemia-reperfusion injury(MIRI),and the late stage of MIRI leads to The rate of heart failure and related complications and death caused by MIRI is increasing,and finding effective ways to intervene in MIRI injury is an urgent clinical problem.Basic experimental studies have shown that myocardial ischemia-reperfusion injury is a disease process jointly regulated by multiple complex classical mechanisms,among which calcium overload,oxidative stress,cell death,and inflammatory response are all considered to be important factors contributing to ischemia-reperfusion injury.The inflammatory response is one of the earliest responses during ischemia-reperfusion injury in the heart,and its process mainly includes the release of inflammatory mediators,infiltration and activation of leukocytes,tissue repair and regeneration.The inflammatory response and oxidative stress injury are closely related,and the occurrence of oxidative stress can trigger the activation of a series of transcription factors.The activation of representative NF-κB transcription factors is accompanied by the release of pro-inflammatory cytokines such as TNF-α,leading to further activation of the inflammatory response and forming a vicious circle to exacerbate tissue injury.Cellular necrosis is another classical pathway in myocardial ischemia-reperfusion injury,and the regulated cell death that occurs with ischemia-reperfusion is likewise related to the regulation of inflammation.Damage associated molecular pattern molecules(DAMPs)are released during cell death,and the accumulation of DAMPs triggers innate immunity and tissue inflammation,contributing to a closed loop of cellular tissue inflammation and regulated cellular necrosis,leading to increasing tissue damage and organ dysfunction.Iron death occurs due to the accumulation of reactive oxygen species that exacerbate lipid peroxidation,which promotes iron deposition leading to cell death releasing damage-related molecular patterns,which in turn activate transcription of the transcription factor NF-κB and induce sterile inflammation.On the other hand,lipid peroxidation can also promote macrophage polarization to activate inflammatory responses.Iron death and inflammatory responses in myocardial ischemia-reperfusion injury accompany each other and are causal,but there are few studies on the synergistic effects of classical pathways on myocardial ischemia-reperfusion injury,and the alterations produced by the synergistic effects are not clear.Based on years of clinical practice and research,our supervisor concluded that the key to the pathogenesis of coronary artery disease is "heat nodulation of blood vessels" and proposed the method of clearing heat and detoxifying toxins to treat coronary artery disease,and created the representative formula HJ11.Preliminary clinical trials have shown that HJ11 can significantly improve the healing status of PCI patients.In this study,we investigated the effect of HJ11 on serum inflammatory factor expression and myocardial pathological injury in rats with ischemia reperfusion(I/R)based on the theory of "heat junction of blood vessels".The results showed that HJ11 not only reduced the expression of inflammatory factors but also improved the oxidative stress injury and mitochondrial structure of myocardial cells in rats,and the preliminary investigation indicated that HJ11 interfered with myocardial ischemia reperfusion injury and iron death.Object:1.To clarify the efficacy of the clinically effective formula HJ11 in the treatment of coronary heart disease,and to investigate the effects of HJ11 on the serological indexes and pathology of rats in the ischemia-reperfusion model by preparing a rat ischemia-reperfusion model and using HJ11 formula for intervention.2.To clarify the possible mechanism of action of HJ11 formula to improve ischemia-reperfusion myocardial injury in rats,and to explore the effect of HJ11 on TLR4/MyD88/NF-κB,a key pathway of iron death and inflammatory response,so as to provide new ideas for the clinical treatment of coronary heart disease.Methods:1.The ameliorative effects of HJ11 on myocardial injury in I/R rats were observed by cardiac ultrasound,ELISA for serological indexes and tissue staining.Cardiac ischemia-reperfusion model was prepared in SD rats,and the ejection fraction and ventricular wall motion indexes by cardiac ultrasound,cardiac enzymes LDH and CK-MB were used as model evaluation indexes,and the morphological and structural changes of myocardium were observed by tissue staining,the white infarct area of myocardium was observed by TTC,and the myocardial changes of I/R rats were observed by Tunel staining.2.ELISA and flow sorting were used to detect the expression of inflammatory factors and the proportion of relevant immune cell subtypes to clarify the regulatory role of HJ11 on the inflammatory response in I/R rats.ELISA was used to detect the expression of inflammatory factors TNF-α,IL-1β and IL-6 in the heart tissue homogenates of I/R rats,and the ratio of each subtype of serum neutrophils and monocytes in rats was detected by flow cytometry.3.ELISA,tissue staining,transmission electron microscopy and protein immunoblotting experiments were used to clarify the intervention effect of HJ11 on iron death in I/R rats.The expression of subferric ions in rat serum and heart tissues was detected by ELISA;the deposition of ferric ions in heart tissues was detected by Prussian blue staining;the structural changes of mitochondria in myocardial tissues were observed by transmission electron microscopy,and the expression of iron death-related proteins was detected by protein immunoblotting assay.4.Small interfering RNA technology and transfection of overexpression plasmids were used to explore the possible mechanism of HJ11’s effect on improving myocardial injury in I/R rats.A cell model of H9c2 ischemia-reperfusion(OGCR)was prepared,and the expression of ACSL4 in H9c2 cells was interfered by transfection of small interfering RNA and overexpression plasmid.The expression of inflammatory factors in cell culture supernatants was observed by ELISA,the nuclear translocation of transcription factor NF-κB was observed by laser confocal,and the expression of TLR4/MyD88/NF-κB and its downstream signaling molecules in cardiomyocytes was detected by protein immunoblotting.Results:1.HJ11 ameliorates myocardial injury in I/R rats.Cardiac ultrasound showed that the left ventricular ejection fraction decreased to 30.67 ± 5.55%in the I/R group,and the ejection fraction increased after intervention with Aspirin and HJ11,and the ejection fraction increased to 47.77 ± 10.28%in the Aspirin group,and the improvement effect was more obvious in the middle and high dose groups than in the low dose group,and the ejection fraction increased to 45.23 ± 5.03%in the high dose group(P<0.05).The results of cardiac enzymes showed that the expression of LDH and CK-MB in the serum of rats in the I/R group increased compared with that in the Sham group,and that the intervention with HJ11 reduced the expression of LDH and CK-B in rats after ischemia-reperfusion,and the effect was the same as that of the positive control drug Aspirin group;the results of TTC staining showed that the expression of white infarct in the I/R group increased compared with that in the sham group.The TTC staining showed an increase in white infarct area in the I/R group compared with the sham-operated group,which was 43.74 ±27.19%(P<0.05),and a significant reduction in white infarct area was seen in the Aspirin and HJ11 interventions,with the most obvious effect in the HJ11-H group,which showed a reduction in white infarct area to 23.85 ± 0.02%,indicating that HJ11 can reduce myocardial ischemia and reperfusion in rats after myocardial reperfusion(P<0.05).HE results showed that HJ11 improved the structural changes of myocardial tissue and reduced inflammatory cell infiltration in I/R rats;TUNEL staining showed that apoptotic cells were significantly increased in the I/R group compared with the sham-operated group,and the positive rate of apoptotic cells in the I/R group was as high as 53.86 ± 6.6%.The positive rate of apoptotic cells in the I/R group was 53.86± 6.78%,and a decrease in apoptotic cells was seen after the intervention with HJ11,with the most significant improvement in the HJ11-H group,where the positive cell rate could be reduced to 18.44 ± 5.17%(P<0.01),indicating that HJ 11 could reduce myocardial cell apoptosis after ischemia-reperfusion in rats.2.HJ11 attenuates the inflammatory response in I/R rats.The ELISA results showed that the expression of each inflammatory factor increased 2-3 times in the model group compared with the sham-operated group for TNF-α,IL-6 and IL-1β,with the expression of TNF-α in the heart tissue of the model group as high as 99.11 ±5.53,IL-6 as 64.60±5.30 and IL-1β as 81.75±10.58.Intervention with HJ11 reduced the rat myocardial tissue homogenates and showed dose-dependent down-regulation,and the expression of inflammatory factors TNF-α was reduced in the HJ11-H group compared with the model group,and the reduction of TNF-α was 56.19±16.58(P<0.01),IL-6 was 38.58±9.37,and IL-1β was 33.64±8.51(P<0.01),indicating that HJ11 can effectively reduce the expression of each inflammatory factor(P<0.01);flow cytometry results showed that HJ11 showed different trends in the regulation of neutrophils and monocytes in the rat I/R model,with increased neutrophil expression seen in the model group compared to the sham-operated group(P<0.01);decreased expression of total monocyte ratio,with increased expression of pro-inflammatory isoforms of monocytes in the model group compared to the sham-operated group.The results of ELISA and flow cytometric sorting indicated that HJ11 had a good inhibitory effect on the inflammatory response after ischemia-reperfusion in rat heart;the fine structure of cardiomyocytes was observed by fluoroscopy,and HJ11 could improve the mitochondrial structure,reduce mitochondrial contraction and mitochondrial cristae breakage after ischemia-reperfusion injury in rat heart.3.HJ11 interferes with Ferrpotosis in I/R rats.The results of ROS showed that the expression of ROS increased in the model group compared with the sham-operated group,and the expression of ROS in the model group could reach 145.5±5.86,which was three times more than that in the sham-operated group 47.00±4.00(P<0.01),and the reduction of ROS was seen in the HJ11 intervention,and the effect was more significant in the HJ11-H group,which could be reduced to 102.70±5.69(P<0.01).The results of MDA and SOD showed that HJ11 reduced the expression of MDA and increased the expression of SOD and GSH in myocardial tissues of rats after myocardial ischemia-reperfusion injury(P<0.05);ELISA detected the expression of subferric ions in serum and myocardial tissues of rats,and the I/R group showed a significant increase compared with the sham-operated group.HJ11 dose-dependently inhibited the deposition of iron ions(P<0.01),and Prussian blue staining results also showed an increase in blue-stained iron ions in the I/R group compared with the normal group,and a decrease in blue-stained area was seen after HJ11 intervention(P<0.05);Western blot detected the expression of iron death key proteins in rat myocardial tissues,and found that HJ11 down-regulated the expression of ACSL4,COX2 and increased the protein expression of GPX4 and FTH-1.The results of repeated in vivo studies in H9c2 rat cardiomyocyte cell lines revealed that HJ11 could effectively reduce oxidative stress injury in H9c2 cells after OGD/R,and could show a dose-dependent reduction in iron ion deposition,reactive oxygen species accumulation,increase H9c2 cell viability and improve myocardial injury after OGD/R;ATP and mtDNA results could indicate that HJ11 could affect ATP and mtDNA results can indicate that HJ11 can affect the energy metabolism of cardiomyocytes and increase the content of ATP and mtDNA(P<0.05);further observation of the fine structure of H9c2 cells by fluoroscopy electron microscopy shows that HJ11 can also improve the structural damage of mitochondria caused by OGD/R and reduce mitochondrial contraction;4.Potential mechanism of action of HJ11 for ameliorating myocardial injury in I/R rats.Western blot results showed that HJ11 upregulated the protein expression of GPX4 and FTH-1,key proteins of iron death(P<0.05),and decreased the expression of ACSL4 and COX2(P<0.05),and HJ11 still exerted an inhibitory effect on iron death in OGD/R injury in cardiomyocytes;the knockdown of ACSL4 in OGD/R-treated H9c2 cells knockdown attenuated iron accumulation,oxidative stress and iron death;the inhibitory effect of HJ11 on OGD/R-triggered iron deposition was reversed by transfection with overexpression of ACSL4 plasmid;the addition of iron death inducer and inhibitor groups revealed that HJ11 could exert an inflammatory effect similar to the efficiency of iron death inhibitor,and it was observed that inflammatory factors were significantly upregulated in the group with the addition of iron death inducer compared with the HJ11 group(The confocal results showed an increase in NF-κB in the nucleus of the OGD/R group compared to the cytoplasm,and the increased NF-κB entry into the nucleus could be reversed after the intervention with HJ11;the protein immunoblotting experiments showed that TLR4,MyD88 and TRAF6 protein expression was upregulated in the OGD/R group compared to the normal group,and NF-κB was reduced in the cytoplasm and increased in the nucleus.The expression of IKBa was not significantly changed,but phosphorylated IKBα was significantly up-regulated(P<0.05);TLR4,MyD88 and TRAF6 protein expression was decreased in the HJ11 intervention group compared to the OGD/R group,and intracytoplasmic NF-κB was increased and intracellular NF-κB was decreased in the nucleus.The expression of IKBa did not change significantly,but the phosphorylated IKBa decreased significantly(P<0.05).The addition of iron death inducer Eartin reversed the effect of HJ11 on the above protein expression,and the effect of Fer-1 on the regulation of protein expression was basically the same in the HJ11 intervention group and the iron death inhibitor group,indicating that HJ11 can exert the effect of iron death inhibitor by reducing the activation of transcription factor NF-κB and regulating the protein expression of TLR4/MyD88/NFκB signaling pathway.It plays a role in reducing the inflammatory response.Conclusion:1.HJ11 attenuates myocardial cytopathic injury caused by myocardial ischemia-reperfusion,improves myocardial function after ischemia-reperfusion injury,reduces the expansion of myocardial infarct area,and decreases myocardial apoptosis in rats;2.HJ11 reduces the release of inflammatory factors after myocardial ischemia-reperfusion and decreases the proportion of neutrophils and monocytes with pro-inflammatory phenotypes,improving myocardial injury from inflammatory response;3.HJ11 side reduces iron ion deposition after I/R injury,attenuates iron death and ameliorates oxidative stress injury caused by ischemia-reperfusion;4.HJ11 plays the role of iron death inhibitor to regulate the expression of ACSL4,a key protein of iron death,to interfere with the development of iron death,and can regulate the changes in the expression of TLR4/MyD88/NF-κB,a key signaling pathway of inflammatory response,to reduce the release of inflammatory factors in ischemia-reperfusion injury of cardiomyocytes and reduce myocardial injury brought by inflammatory response,in conclusion,HJ11 can reduce myocardial injury brought by ischemia-reperfusion injury by regulating ACSL4-mediated iron death and TLR4/MyD88/NF-κB to inhibit myocardial I/R injury. |
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