Protective Effect And Mechanism Of Wedelolactone On Acute Heat Stress Injury Of Primary Myocardial Cells

Heat stress can cause a series of physiological reactions and metabolic changes in broilers to affect the normal growth of the body,and even lead to death.Studies have shown that broilers under heat stress will bring higher pumping load and pressure to the heart,causing significant damage to the heart tissue.Wedelolactone,as a coumarin compound,has a wide range of biologically active functions.Studies have shown that Wedelolactone has a protective effect on myocardial cells,but it has not been reported that alleviated heat stress damage.Therefore,this experiment was to extract primary myocardial cells from Wenchang chicken embryos.The optimal temperature and time of acute heat stress were chosen to construct an in vitro model of acute heat stress in primary myocardial cells of Wenchang chicken.To explore the effect of Wedelolactone on myocardial cells against acute heat stress.The activity of primary myocardial cells and the changes of LDH and AST enzyme activities were analyzed by adding different concentrations of Wedelolactone.By verifying the effect of Wedelolactone on the signal pathway of NF-?B and MAPK protein in myocardial cells,the molecular mechanism of its anti-acute heat stress effect was analyzed.The experimental research results are as follows:1 With the increase of temperature and time of acute heat stress treatment,the activity of chicken primary myocardial cells of Wenchang chicken gradually decreased,and morphological damages occurred in different degrees.After treatment at 45 ? for 4 hours,the survival rate of primary myocardial cells decreased significantly(p<0.01),and the cell survival rate was only 79.70 ± 5.61% of normal conditions.The effect of acute heat stress injury is obvious.2 The Wedelolactone at room temperature is non-toxic to primary myocardial cells and has a cell-activating effect,and the effect is concentration-dependent.At a concentration of 80 ?M,the cell survival rate could be significantly increased by 1.3 times(p<0.01).3 Under acute heat stress conditions,the concentration of Wedelolactone 5?80 ?M could significantly improve the survival rate of primary myocardial cells,and down-regulate the increase of LDH and AST enzyme contents caused by acute heat stress.It shows concentration dependence,especially when the concentration is 80 ?M(p<0.01).At the same time,there was no significant difference in the LDH and AST enzyme activities of different concentrations of Wedelolactone in the normal temperature control group(p>0.05).4 Under acute heat stress,the relative expression of p65,p-p65 and p-I?B? in primary myocardial cells increased significantly.Increased levels of p65 and I?B? phosphorylation indicate activation of the NF-?B signaling pathway.However,the addition of Wedelolactone could significantly reduce the relative expression of p-p65 and p-I?B?.Inhibition of p65 and I?B? phosphorylation level,inhibition of NF-?B signaling pathway activation,showing a concentration-dependent inhibition effect at the concentration of20?80 ?M is the most obvious.5 Acute heat stress significantly increased the relative expression of p38,p-p38,JNK,p-JNK and p-ERK in primary myocardial cells.Increasing the phosphorylation of p38,JNK and ERK proteins activates the MAPK signaling pathway.However,Wedelolactone could down-regulate the expression of p38 and JNK,and effectively reduce the phosphorylation of p38,JNK and ERK protein,inhibit the activation of MAPK signaling pathway,showing a concentration-dependent inhibition effect at the concentration of20?80 ?M.In summary,the appropriate concentration of Wedelolactone is non-toxic to primary myocardial cells and has a certain promoting effect,which could effectively reduce the damage of acute heat stress on myocardial cells.The optimal concentration is 80 ?M.The mechanism of action is related to the ability of Wedelolactone to inhibit the activation of NF-?B and MAPK protein signaling pathways in myocardial cells due to acute heat stress.In this experiment,by elucidating the effect and mechanism of the heat stress effect of the Wedelolactone,it provides a new idea for reducing the heat stress damage of broilers,and provides the theoretical basis and scientific basis for the subsequent further research and development of the Wedelolactone as a medicinal feed additive for broilers.