| PARTⅠESTABLISHMENT AND EVALUATION OF CARDIAC HYPERTROPHY MODEL INDUCED BY ISOPROTERENOL IN RATSObjective:To established and evaluated animal model of isoproterenol induced pathological cardiac hypertrophy(PCH).Methods:A rat model of PCH was established by subcutaneous injection of isoproterenol(ISO).Twenty 8-week-old male SD rats were randomly divided into the normal control group(Control group)and isoproterenol(5mg/kg/d)injection group(ISO group).After 14 consecutive days of intervention,each rat was examined by ultrasound using The Vevo~?3100 LT Imaging System.The myocardial tissues of the two groups of rats were collected after sacrifice and stored for further detection after weighing.Hematoxylin-eosin(HE)staining was performed on the myocardial tissue to understand the pathological changes of myocardial tissue,and Masson staining and Sirius red staining were performed to evaluate the myocardial fibrosis.The protein and m RNA expression levels of myocardial hypertrophy markers(ANP,BNP,andβ-MHC)were detected by Western blot and RT-PCR.Results:(1)Compared with the Control group,the LVPWd(1.610±0.122mm vs.2.227±0.142mm),LVPWs(2.447±0.134mm vs.3.435±0.143mm),LVAWd(1.328±0.159mm vs.2.225±0.114mm)and LVAWs(2.163 0.119mm vs.3.634 0.187mm)in ISO group increased significantly(P<0.001),while LVIDd(8.037±0.258mm vs.6.692±0.317mm),LVIDs(5.304±0.213 vs.4.174±0.248)were significantly lower than those in the Control group(P<0.001);The ISO group had HW/BW of 3.633±0.220 and LVW/BW of 2.015±0.112,which were significantly higher than those in the Control group(HW/BW:2.867±0.125,LVW/BW:1.547±0.100)(P<0.001);(2)Compared with the Control group,rats in the ISO group had disordered myocardial structure arrangement,interstitial edema,widened gap,karyolysis or karyopycnosis,myocardial fiber rupture,increased distribution of collagen fibers,and obvious fibrosis around blood vessels;WGA staining showed that the cross-sectional area of myocardial cells in the ISO group was(331.09±22.77μm~2)significantly higher than that in the Control group(199.39±19.12μm~2)(P<0.001);(3)Compared with the Control group,the protein expression levels of ANP,BNP,andβ-MHC in the ISO group were significantly increased(P<0.001);RT-PCR showed that the m RNA expression levels of ANP,BNP,andβ-MHC in the ISO group were significantly higher than those in the Control group(P<0.001).Conclusion:The rat model of PCH was successfully induced by subcutaneous injection of ISO(5 mg/kg/d)for 14 days.PART Ⅱ EFFECT AND POSSIBLE MECHANISM OF LUTEOLIN ON ISOPROTERENOL INDUCED CARDIAC HYPERTROPHY IN RATSObjective:To explore whether Luteolin has a protective effect on isoproterenol induced cardiac hypertrophy in rats and the possible mechanism.Methods:Thirty 8-week-old male SD rats were randomly divided into three groups: normal control group(Control group)(n=10),ISO group(n=10),and ISO+Luteolin group(n=10).The rat model of cardiac hypertrophy was induced by subcutaneous injection of ISO using the method in Part 1.Three groups of rat were examined by ultrasound using The Vevo? 3100 Micro-ultrasound Imaging System.The hearts of rats in each group were collected after sacrifice.The myocardial tissues of rats in each group were subjected to HE staining for evaluation of pathological changes in myocardial tissue,Masson staining and Sirius red staining for evaluation of myocardial fibrosis,transmission electron microscopy for detection of ultrastructural changes in myocardial cells,CD68 immunohistochemical staining for evaluation of inflammatory cell infiltration in myocardial tissue,DHE staining for evaluation of reactive oxygen levels in myocardial tissue,and WGA staining for evaluation of the cross-sectional area of myocardial cells.The levels of IL-1β,IL-6,and TNF-α in the myocardial tissue of rats were detected by ELISA,the LDH level,MDA level and SOD activity of myocardial tissue were detected to assess the oxidative stress level.The protein expression levels of ANP,BNP,β-MHC,AMPK,p-AMPK,and HO-1 were detected by Western blot.The m RNA expression levels of ANP,BNP,β-MHC were detected by RT-PCR.Results :(1)The LVPWd(2.186±0.412 mm vs.2.627±0.260mm),LVPWs(2.859±0.337 mm vs.3.622±0.413mm),LVAWd(1.785±0.178 mm vs.2.187±0.299mm),LVAWs(2.962±0.343 mm vs.3.469±0.408mm)of ISO+Luteolin group was significantly lower than those in the ISO group(P<0.05),while LVIDd(6.643±0.441 mm vs.5.971±0.399mm)and LVIDs(3.629±0.571 mm vs.2.792±0.543mm)were significantly higher than in the ISO group(P<0.05);The HW/BW(2.726±0.118 vs.3.651±0.112)and LVW/BW(1.691±0.099 vs.2.067±0.060)of the ISO+Luteolin group were significantly lower than those in the ISO group(P<0.001);(2)The degradation of cardiomyocytes,interstitial edema,necrosis and dissolution of cardiomyocytes were alleviated in the ISO + luteolin group compared with the ISO group,while the myocardial structure was largely preserved and the distribution of myocardial muscle bundles was more regular;The extent of interstitial fibrosis in the myocardial tissue of rats in the ISO + luteolin group was significantly reduced compared with that in the ISO group,and perivascular collagen fibers were reduced;(3)Compared with the ISO group,the cross-sectional area of myocardial cells in the ISO+Luteolin group(205.95±30.18μm2)was significantly lower than that in the ISO group(317.15±30.58μm2)(P<0.001),the protein and m RNA expression levels of ANP,BNP,and β-MHC in the ISO+Luteolin group were significantly reduced(P<0.001);(4)Immunohistochemical staining showed that CD68+ macrophage infiltration in myocardial tissue was reduced in the ISO+luteolin group compared with the ISO group;The myocardial tissue levels and m RNA expression of IL-1β,IL-6,TNF-α in the ISO+Luteolin group were significantly lower than those in the ISO group(P<0.001);(5)DHE staining showed that ROS production in myocardial tissue of ISO+Luteolin group was significantly lower than that of ISO group(P<0.001);Compared with the ISO group,rats in the ISO+Luteolin group had decreased LDH and MDA levels(P<0.001)and increased SOD activity(P<0.001);(6)In the ISO group,the protein expression levels of p-AMPK and HO-1 were significantly decreased compared with the Control group(P<0.001),whereas was significantly increased after Luteolin intervention(P < 0.001).Conclusion:Luteolin reduced ISO-induced PCH in rats,alleviated inflammatory reactions,maintained oxygen reduction balance,and upregulated the expressions of p-AMPK and HO-1.PART Ⅲ LUTEOLIN ATTENUATES ISOPROTERENOL INDUCED H9C2 CELLS HYPERTROPHY VIA AMPK / HO-1 PATHWAYObjective : To investigate the effect and possible mechanism of luteolin on ISO-induced hypertrophy of H9c2 cells.Methods:H9c2 cells were used to establish an in vitro model,and isoproterenol(10μM)was used to stimulate H9c2 cells to establish a hypertrophy model.There are two grouping methods in this part.The CCK-8 experiment was conducted to determine the optimal concentration of luteolin.Three groups were set up: the normal Control group(Control group),cell model group(ISO group),and luteolin treatment group(ISO+Luteolin group).The cross-sectional areas of H9c2 cells in each treatment group were detected with the Actin-Tracker Green-488 probe.The protein and m RNA expression levels of ANP,BNP,and β-MHC in cells of each group were detected by Western blot and RT-PCR.The levels of IL-1β,IL-6,TNF-α,and m RNA expressions in the medium and cells were detected by ELISA and RT-PCR,respectively.The DCFH-DA probe was used to detect the ROS level in cells of each group,detect the intracellular MDA activity and SOD activity,and detect the LDH level in the culture medium.The protein expression levels of AMPK,p-AMPK,and HO-1 in the cells of each treatment group were detected by Western blot.The CCK-8 experiment was used to determine the optimal concentration of AMPK inhibitors Compound C and HO-1 inhibitor Zn PPIX.Five groups were set up: the normal Control group(Control group),the cell model group(ISO group),the luteolin treatment group(ISO+Luteolin group),and the ISO+Luteolin+Compound C group,and the ISO+Luteolin+Zn PPIX group.The above-mentioned experiments and assays were conducted.Results:(1)The cross-sectional area of H9c2 cells in the ISO group was(3808.15±411.18μm2)significantly higher than that in the Control group(1730.52±278.30μm2)(P<0.001),while that of the ISO+Luteolin group was(2147.21±362.93μm2)significantly lower compared with the Control group(P<0.001);the expression levels of ANP,BNP,and β-MHC protein and m RNA in the ISO group were significantly increased compared with the Control group(P<0.001),while the protein and m RNA expression levels of ANP,BNP,and β-MHC in the ISO+Luteolin group were significantly decreased compared with the ISO group(P<0.001);(2)The levels of IL-1β,IL-6,TNF-α and m RNA expressions in the ISO group were significantly higher than those in the Control group(P<0.001),while the levels of the above three inflammatory factors were inhibited by Luteolin treatment(P<0.001);(3)Compared with the Control group,the intracellular ROS fluorescence intensity,LDH content in the medium supernatant and intracellular MDA in the ISO group were significantly increased(P<0.001), and the cells in the ISO+Luteolin group were significantly decreased(P<0.001);The intracellular SOD in the ISO group was significantly lower than that in the Control group(P < 0.001),while that in the ISO+Luteolin group was significantly higher than that in the ISO group(P<0.001);(4)The protein expression levels of p-AMPK and HO-1 in the ISO group were significantly lower than those in the Control group(P < 0.001),while the expression levels of p-AMPK and HO-1 proteins in the ISO+Luteolin group were significantly higher than those in the ISO group(P<0.001);(5)The cross-sectional area of cells and the expression levels of ANP,BNP,β-MHC protein and m RNA in the ISO+Luteolin+Compound C group and the ISO+Luteolin+Zn PPIX group were significantly higher than those in the ISO+Luteolin group(P<0.001);(6)The levels and m RNA expressions of inflammatory factors(IL-1β,IL-6,TNF-α)were significantly higher than those in the ISO+Luteolin group(P<0.001);(7)ROS fluorescence intensity,LDH content in culture medium supernatant and intracellular MDA content were significantly higher than those in the ISO+Luteolin group(P<0.001),while SOD content was significantly lower than that in the ISO+Luteolin group(P<0.001);(8)The protein expression levels of pAMPK in the ISO+Luteolin+Compound C group and and HO-1 the ISO+Luteolin+Zn PPIX group were significantly lower than those in the ISO+Luteolin group(P<0.001).Conclusion : Luteolin may alleviate PCH by attenuating inflammatory response and oxidative stress in ISO-induced H9c2 cells via activating AMPK / HO-1 signaling pathway. |
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