The Protecting Effects Of Betulin On Impaired Myocardial Cells

Myocardial ischemia-reperfusion injury (MIRI) means myocardial blood flow interruption or after the period of time decrease then the blood flow recover, cause the phenomenon of metabolism, structural damage aggravate. It’s pathogenetic mechanism is complex, including oxygen radical, calcium overload, inflammatory response, apoptosis and mitochondrial dysfunction and so on, and different paths form a network interaction by a variety of cytokines, including aggravate cell damage, In recent years, myocardial inflammation attract more and more attention in the process of myocardial ischemia-reperfusion injury. Many factors or stress can make expression of myocardial and excreted by inflammatory cytokines, like tumor necrosis factor-a (TNF-a), interleukin1(IL-1), interleukin6(IL-6), monocyte chemoattractant protein-1(MCP-1), cell adhesion molecules-1(ICAM-1) and so on, these cytokines usually cause NF-κB activation of signal pathways.Nuclear factor κB (NF-κB) is inflammation of the mammalian cells regulatory factors. In the process of inflammation, NF-κB activated from the cytoplasm translocate to the nucleus and binding DNA specificity, which in turn induce its downstream a variety of gene expression of inflammatory mediators, which lead to the aggravate of the inflammation, increase myocardial injury. So, NF-κB signal transduction pathway’s excitation has important meaning to the modulation of the myocardial cell inflammatory response. Signal transduction and transcriptional activation factor3(STAT3) widely participate in cellular stress, growth, proliferation, differentiation, poptose and many other biological effect. Several studies show that, STAT3participate in a variety of heart physiology, pathology activities, like myocardial cell survival, myocardial revascularization, mitochondrial energy metabolism, changes of extracellular matrix and inflammatory response. Short of STAT3will cause the myocardial cell inflammation and myocardial fibrosis, increase the occurrence of heart failure.Betulin is a kind of important pharmacological substances extract from birch bark, and can change to betulinic acid, and have many biological activity like anti-inflammatory, antibiosis, antiviral, antineoplastic and anti-HIV function, aroused people’s great research interest. Recent studies also found that betulin can inhibit cholesterol regulating element binding protein (SREBP) and reduce cholesterol and three acyl glycerin, improve the obese patients with hyperlipidemia and insulin resistance, and has obvious treatment for type2diabetes. But at this moment, the influence of betulin in myocardial cell injury protection is still lack of research.The purpose of this study was to investigate betulin protection of myocardial cell injury and its signal mechanism. Given that inflammation plays an important part in myocardial ischemia-reperfusion injury, the experiment mainly concentrated in two parts of study. Firstly, we constructed perfused rat heart model to study the effect of betulin pretreatment on myocardial ischemia-reperfusion injury, and then focused on anthropogenic AC16myocardial cells as experimental object, from the view point of the inflammatory response to explore its protective effects on myocardial cell and molecular signal mechanisms.PART ⅠThe effect of betulin in resisting myocardial cells inflammationObjectiveTo explore the resistance effects of betulin preconditioning on myocardial cell inflammation in rats.Methods50rats were randomly divided into five groups, each with10rats. Quickly dissociate the heart after anesthesia, placed it in the Langendorff shelves, perfused through the aorta cannulation.①alse ischemia/reperfusion group (Sham):Hearts perfused with K-H solution for120min.②ischemia/reperfusion model group (Model):Hearts perfused with KH solution for50min, stopping irrigation30min, and then reperfused with K-H solution for40min.③three different concentrations of betulin (Betulin:25/50/100mg/L) preconditioning group:After using the K-H solution continuous heart perfusing for10min, and separately using contain corresponding concentration of betulin’s K-H solution perfusing for5min, and then using the K-H solution without betulin perfusing for5min, repeat like this, total for four times, and then handle the same Model group.After perfusion, cut out a small piece of myocardial tissue from the apical portion, and then put it in10%paraformaldehyde solution in a refrigerator, at4℃, and then we proceed the myocardial tissue pathology HE staining, TTC staining measure, TUNEL method to detect the cell apoptosis and immunohistochemical determine TNF-a, ICAM-1and NF-κB expression level; picked the left most of the left ventricular myocardium and eliminated the connective tissue, made it into myocardial homogenate, put it-20℃situation to store, and used the corresponding kit to detect the biochemical criterion like LDH, CK and MPO vitality.Results1. CK vitality in Model group’s rats myocardial tissues is14.29±2.08kU/g protein, LDH vitality is1027±66U/g protein, decrease compare to the Sham group (P<0.01); Betulin group can concentration-dependent inhibit post I/R damage CK and LDH in myocardial tissue of rats leak to the blood, the effect is more apparent in Betulin100mg/L group, CK and LDH activity increase than the Model group (P<0.01), which is22.42±1.44kU/g protein and1691±79U/g protein.2. Compare with the Sham group, Model group rats sufferred a large area of myocardial infarction, after Betulin100mg/L pretreatment the myocardial injury reduce, the area of myocardial infarction (IS/AAR%=18.54±4.92) significantly reduced than the Model group (IS/AAR%=42.13±6.27),(P<0.01).3. Myocardium HE staining can see microscopic Model group’s rats myocardial fibers has obvious corrugated change and myocardial contraction bands, part of the fiber breakage and dissolute; Betulin group’s damage change is lesser, myofibril arranged almost neat, sarcomere almost complete, without characteristic changes like shrinkage and corrugated change.4. Compare with Sham, Model group’s TUNEL stain is the most obvious, and after the pretreatment of betulin, TUNEL’s staining intensity show dose dependent decrease, which indicates that betulin can significantly inhibit myocardial I/R to cause the cell apoptosis.5. Model group’s inflammatory factor NF-κB, TNF-a and ICAM-1’s expression increase obviously, the average grey value of positive reaction increased significantly than the Sham group (P<0.01). Compare with the Model group, Betulin group’s expression of NF-κB, TNF-a and ICAM-1all decrease,100mg/L group’s effect is more obviously (P<0.01).6. MPO vitality is very low in myocardial tissue homogenate of Sham group’s rats (0.15±0.007U/g protein), but Model group’s MPO vitality (0.87±0.012U/g protein) increase than the Sham group (P<0.01); but the MPO vitality in all Betulin group’s cardiac muscle tissue is lower than the model group (P<0.05).ConclusionsBetulin preprocessing can reduce myocardial cell inflammation in rats and protect myocardial tissue ischemia-reperfusion injury. PART ⅡResearch of the molecular mechanism of betulin developing myocardial cell inflammationObjectiveTo explore the molecular mechanism of betulin developing myocardial cell inflammatory effect.MethodsAC16cells were purchased from ATCC which were derived from adult ventricular muscle cells, culturing by the DMEM medium. Bottled after cell growth and fusion, centrifugal to get cell suspension, put partial shipment in culture plates. When the cell density reached to70%-80%and proceed grouping experimental treatment, and collect cell supernatant and cell, cryopreserved store and check in-80℃refrigerator.1. After giving the blank control group betulin for4hours, adding TNF-a, collect the cell supernatant, detect the IL-6and MCP-1protein concentration by EILSA testing, collect the cell RNA and then reverse transcription to proceed Real-time PCR to detect the expression of inflammation gene such as IL-6, MCP-1and IL-1β.2. Transfect NF-κB reporting gene plasmid in AC16cell, and give the stimulating of TNF-a and betulin. Using luciferase report and p65anti-body ChIP experiment to analyze the gene expression of NF-κB signal transduction, and evaluate the influence of betulin to the transcriptional activity of NF-κB in AC16cell.3. After incubate AC16cell by Betulin, detect STAT3’s phosphorylation and the level of it’s downstream target genes like SOCS3and BCL-xL.4. By adding STAT3pathway’s inhibitor AG490and micro RNA interference fragment, detect whether the STAT3pathway is necessary for the anti-inflammatory effect of betulin.Results1. TNF-a can significantly improve AC16cell’s mRNA expression level of IL-6, MCP-1and IL-1β, but betulin can significant inhibit TNF-a induced inflammation gene’s expression, the cell’s protein concentration of IL-6and MCP-1are all lower than the control group.2. TNF-a can significantly activated NF-κB reporting gene plasmid’s transcriptional activity, promote P65’s accumulation, phosphorylation and acetylation in nucleus, but betulin can significant inhibit TNF-a’s effect, block NF-κB signal’s excitation in AC16cells.3. After betulin incubating AC16cell, the cell’s STAT3phosphorylation enhance, the expression of the downstream gene like SOCS3and BCL-xL enhance, confirm the process has STAT3’s pathways activating.4. After giving STAT3inhibitor-AG490, betulin’s expressing inhibitory effect of proinflammatory cytokine IL-6and MCP-1effect decrease; Using micro RNA interference fragment to knockout the cell endogenous STAT3expression, betulin’s protecting effect reverse, confirm to indicate that betulin’s cardioprotective effects rely on STAT3pathway’s activation.ConclusionsBetulin activate the STAT3signal pathway, and inhibit the activation of myocardial cell inflammatory pathways and the expression of the inflammation related gene.