| Objective This topic is aim to observe the effects of LGZGD on IKK/I?B/NF-?B signaling pathway and its target moleculars in myocardial, and explore its intervention mechanism of ventricular remodeling.Methods In vivo, coronary artery ligation method is used to copy the ventricular remodeling after myocardial infarction model in 120 SD rats(?,body weight 180±20g).After 2 weeks, these rats were divided randomly into model group, LGZGD(2.1?4.2?8.4 g/kg) groups, Captopril group(0.00257 g/kg), Stilbene Li cardiac capsules group(0.3702 g/kg)and setting sham-operation group. Rats in treating groups were given medicines by intragastric(10ml/kg) for 4 weeks, while rats of model group and sham-operation group were given the same volume of distilled water instead.Weigh these rats once a week. 30 min later after the last administration, under aseptic conditions the rats heart were separated and blood were collected by abdominal aortic method. HE staining method and Masson staining were used to observe the changes of myocardial histopathological and collagen fibers in rats respectively; We use ELISA method to detect the contents of TNF-??IL-6 and IL-1? in rat serum of each group; Western blot was performed to analyze the expression of NF-?Bp65?IKK-??I?B-? and p-I?B?.In vitro, cardiomyocytes were isolated and cultured from newborn SD rats by trypsin digestion method, and these cardiomyocytes were identified by fluorescence immunoassay. Cardiomyocytes in good state were divided into 6 groups randomly:normal group, LPS group, blank serum group(20%),serum(5%?10%?20%)containing LGZGD(8.4 g/kg)groups and the cell density of cardiocytes was 3×105.Then cardiomyocytes were incubated with normal rat serum(20%)and serum containing LGZGD(5%?10%?20%) respectively for 12 hours, then LPS( 100ng/m L)was added into each group except normal group for another 12 hours, aim to establish the injury model of cardiomyocytes. The contents of TNF-??IL-1? and IL-6 in cardiomyocytes were detected by ELISA; Western blot was employed to detect the expression of NF-?Bp65?IKK-??I?B-? and p-I?B? in the cytoplasm and NF-?Bp65 in the nucleus.Results In vivo,(1) the observation of myocardial tissue morphology: HE staining shows that the myocardial tissue of rats in sham-operation group stain evenly and with regular cell shape, the structure is clear, cells arrange compact and orderliness,myocardial fibers direction is consistent and neat, a few of fibroblasts are seen.While the myocardial tissue of rats in model group stained lightly, the myocardial fibers are broken and arrange in disorder like wavy, the cell morphology is fuzzy, cardiomyocytes sarcoplasmic dissolve, microvascular and red blood cells are seen scatteredly,fibroblasts increase accompanied by inflammatory cells infiltration; Compared with model group, the pathological characteristics of rats are improved in each treatment group, myocardial fibers arrange more neatly, the nuclear shape tend to be more clear,fibroblasts reduce with only a small amount of inflammatory cells infiltration. Masson staining shows myocardial cells were dyed red, and the collagen fiber blue. The myocardial cells in sham-operation group arrange orderly and tidy, a few collagen fibers are seen in myocardial matrix and uniformly distributed; in model group collagen fibers in myocardial matrix increase obviously and distribute unevenly, myocardial cells arrange in disorder; myocardial interstitial fibers are decreased in each treating group and cardiac muscle tissues are improved.(2) The effects on the content of inflammatory cytokines in rats serum: ELISA shows that the contents of TNF-??IL-6 and IL-1? in model group significantly increase compared with sham-operation group, there are significant differences between them(P<0.01); The contents of TNF-??IL-6 and IL-1? in each treatment group reduce significantly compared with model group, which shows significant differences(P<0.05,P< 0.01), and the contents of IL-1? and IL-6 gradually reduce with the increase of the dose of LGZGD, which shows dose-dependent manner.(3) The effects on the expression of NF-?Bp65?IKK-??I?B-? and p-I?B? in myocardial tissue: western blot shows that significant increases of NF-?Bp65?IKK-? and p-I?B? expression, decrease of I?B-? expression are observed in model group compared with sham-operation group, and there are remarkable difference between them(P<0.01). The NF-?Bp65?IKK-? and p-I?B? proteins in each treatment group are significantly lower and the I?B-? protein is significantly higher than that in model group, which shows remarkable difference(P<0.05, P<0.01). We also observe that with the increase of the dose of LGZGD, the expression of IKK-? and p-I?B? are down regulated gradually, which is dose-dependent.In vitro(1) The identification of myocardial cells: fluorescence immunoassay shows myocardial cell cytoplasm is dyed red, and the negative cells no red cytoplasm. The positive rate of myocardial cells is more than 94%.(2) The effects on the content of inflammatory cytokines in myocardial cells: ELISA shows that the contents of TNF-??IL-6 and IL-1? in LPS group elevate significantly compared with normal group, and there are significant difference between the two groups(P<0.01); Compared with LPS group, the contents of TNF-??IL-6 and IL-1? in blank serum group have no significant differences(P>0.05), while that in serum(5%?10%?20%)containing LGZGD groups all decline markedly(P< 0.05,P< 0.01),and show a trend of dose dependent.(3) The effects on the expression of NF-?Bp65?IKK-??I?B-? and p-I?B? in the cytoplasm and NF-?Bp65 in the nucleus: western blot shows that the expression of NF-?Bp65?IKK-??p-I?B? in the cytoplasm and NF-?Bp65 in the nucleus increase obviously, the expression of I?B-? is dramatically down-regulated in LPS group compared with normal group, presenting a significant difference between them(P<0.01); Compared with LPS group, the expression of NF-?Bp65?IKK-??p-I?B? in the cytoplasm and NF-?Bp65 in the nucleus in serum(5%?10%?20%)containing LGZGD groups significantly lower(P<0.05,P<0.01),while significant up-regulation of I?B-? expression is observed(P< 0.01),and all seem to be dose-dependent.Conclusion LGZGD can improve myocardial tissue pathologic morphology and myocardial tissue fibrosis in rats after myocardial infarction, and its mechanism may be related to the inhibition of the activation of IKK/I?B/NF-?B signaling pathway, further inhibiting the overexpression of myocardial inflammatory cytokines TNF-??IL-6 and IL-1?. |
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