Effect Of Medicated Serum Containing Bawei Chenxiang Powder On Inflammatory Response Of The Cardiomyocyte Injured By Hypoxia/Reoxygenation

Objectives: In this study,we established an in vitro hypoxia/reoxygenation injury model for H9c2 cardiomyocytes,combined with serum pharmacological methods,to investigate the protective effect of Bawei Chenxiang powder containing serum on hypoxia/reoxygenation injury in H9c2 cells,and to investigate the effect of Bawei Chenxiang powder containing serum on the inflammatory response of hypoxia/reoxygenation injured cardiomyocytes.Methods:The effect of different concentrations of blank serum addition(0%,10%,20%,30%,40% and 50%)on the viability of normal cultured H9c2 cardiomyocytes was detected by MTT method,which was used to determine the working concentration of suitable drug-containing serum.H9c2 cardiomyocytes were treated with hypoxia in a triple-air incubator for 8 h,and then replaced with fresh high-sugar medium for 4 h of conventional culture to establish a hypoxia/reoxygenation model.The experimental groups were divided into blank group,hypoxia/reoxygenation model group,low,medium and high dose groups of Bawei Chenxiang Powder serum and positive control(compound Danshen tablets)group.Blank group and model group were added with blank serum,adding drug-containing serum to the drug group.Then treated with hypoxia/reoxygenation(hypoxia 8 h/reoxygenation 4 h),after the intervention was finished.Inverted microscope was used to observe the growth status and morphological changes of H9c2 cardiomyocytes after hypoxia/reoxygenation treatment.The survival rate of H9c2 cardiomyocytes after hypoxia/reoxygenation treatment was detected by CCK-8 method.Activity of LDH and CK and Levels of inflammatory factors IL-1,IL-6,IL-8,COX and TNF-α in the cell supernatant after hypoxia/reoxygenation treatment were measured by spectrophotometry or Elisa.The expression of IL-6,TNF-α,NF-κB,MPO and SIRT1 m RNA and the protein expression of NF-κB,MPO and SIRT1 in H9c2 cardiomyocytes after treated with hypoxia/reoxygenation were detected by real-time fluorescence quantitative PCR(QPCR)and Western blotting(WB).Results:1.The survival rate of H9c2 cells in the 20% blank serum group was better compared with H9c2 cells cultured with 0%,10%,30%,40% and 50% blank serum,and the difference was statistically significant(P<0.05).2.Compared with the cells in the normal group,the cells in the model group showed cell membrane rupture and cell atrophy,and the cell survival rate was significantly lower(P<0.05);compared with the cells in the model group,the myocardial cells in the low,medium and high dose groups of Bawei Chenxiang Powder containing serum were relatively intact,and the cell survival rate was significantly higher(P<0.05),and the cell survival rate in the medium dose group was the highest.3.After hypoxia/reoxygenation,compared to the blank group,significantly higher expression of LDH,CK and inflammatory factors IL-1,IL-6,IL-8,COX and TNF-αin the supernatant of the model group(P<0.05);compared with the model group,the expression of LDH and CK was significantly lower(P<0.05)in the middle and high dose groups of Bawei Chenxiang Powder containing serum,while the expression of inflammatory factors IL-1,IL-6,IL-8,COX and TNF-α were significantly lower(P<0.05)in the low,middle and high dose groups of Bawei Chenxiang Powder containing serum.4.Compared with the blank group,IL-6,TNF-α,NF-κB and MPO m RNA expression were significantly increased(P<0.05),SIRT1 m RNA expression was significantly decreased(P<0.05),MPO and NF-κB protein expression was significantly increased and SIRT1 protein expression was significantly decreased(P<0.05)in the model group;compared with the model group,the relative expression of IL-6,TNF-α,NF-κB,and MPO m RNA was significantly lower(P<0.05),the relative expression of SIRT1 m RNA was significantly increased(P<0.05),the expression of MPO and NF-κB protein was significantly lower,and the expression of SIRT1 protein was significantly higher in the Bawei Chenxiang Powder containing serum group,all of which were statistically different(P< 0.05)Conclusion: Medicated serum containing Bawei Chenxiang Powder has a protective effect against hypoxia/reoxygenation injury in H9c2 cardiomyocytes and inhibits the myocardial inflammatory response to hypoxia/reoxygenation injury.The mechanism may be exerted by inhibiting the NF-κB inflammatory signaling pathway and reducing the levels of inflammatory factors IL-6 and TNF-α.