| Objective To investigate the relationship between myocardial fibrosis and toll-like receptor 4(TLR4)/ nuclear factor-κB(NF-κB)signaling pathway in septic rats and the intervention effect of ammonium pyrrolidine dithioformate(PDTC)on lipopolysaccharide stimulated myocardial fibrosis in rats.Methods 30 healthy SD rats were randomly divided into CONTROL group(n=6),PDTC+LPS group(n= 12)and LPS group(n= 12)by random number table.Twenty-four hours after intraperitoneal injection of LPS,5 ml of arterial blood was collected from the aorta of the rats and centrifuged.The levels of inflammatory cytokines and troponin in the supernatant were detected by ELISA.The morphological changes of myocardial tissue were observed under HE staining microscope.The expression and nuclear translocation of NF-κB p65 in myocardial tissue of each group were detected by IHC.Masson staining was used to observe collagen deposition in rat myocardium.The levels of IL-6,TNF-α,NF-κB p65 and phosphorylation(p-NF-κB p65)in myocardial tissue were detected by Western blotting.After the resuscitation of rat primary myocardial fibroblasts,the cells were cultured at a ratio of 1∶3 until the adherent cells in the visual field were more than 90% under the microscope.Cell counting kit8(CCK-8)was used to detect the proliferative activity of the 3rd generation cells under different concentrations of lipopolysaccharide and PDTC.The third generation cells were divided into control group,lipopolysaccharide group and PDTC group.The protein expression levels of TLR4,NF-κB-p65,phosphorylated NF-κB-p65(p-NF-κB-p65)and transforming growth factor β1(TGF-β1)in rat myocardial fibroblasts were detected by western blotting.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect the m RNA relative expression levels of type Ⅰ collagen and TGF-β1.Results Compared with the results in the Control group,WB results in the LPS group showed that the activation of NF-κB p65 in the myocardial tissue of rats was significantly increased(p-NF-κB p65/NF-κB p65 ratio: 1.34±0.15 vs 1.00±0.00,P < 0.05)and TNF-αprotein expression increased after homogenization(IL-6:1.42 ±0.15 vs 1.14±0.07,TNF-α :1.23±0.10 vs 1.00±0.04,both P < 0.05).The results of immunohistochemistry showed that the positive expression of NF-κB p65 in the nucleus of cardiomyocytes was significantly increased.The results of HE showed that the LPS group had interstitial edema,infiltration of inflammatory cells in different degrees,and breakage of muscle fibers in myocardial tissue.The results of ELISA showed that the levels of IL-6,TNF-α and c Tn I in serum were significantly increased,IL-6(Pg/ml): 16.66±5.28 vs.4.52± 1.80,TNF-α(Pg/ml):20.16±3.88 vs.7.13±0.83,c Tn I(Pg/ml): 79.01±17.25 vs.54.92± 16.08,both P<0.05.Compared with LPS group,PDTC+LPS group:(1)WB: the phosphorylation level of NF-κB p65 in myocardial tissue was decreased(p-Nf-κb p65/NF-κB p65 ratio: 1.34±0.15 vs.0.92±0.07,P < 0.05);The protein expression of IL-6 and TNF-α decreased(IL-6/GAPDH): 1.42±0.15 vs 1.14±0.07,(TNF-α/GAPDH): 1.23±0.10 vs 1.00±0.04,both P< 0.05.(2)IHC: the positive expression of NF-κB p65 in myocardial nucleus was significantly reduced.(3)HE: myocardial edema,hemorrhage,inflammatory cell infiltration and myocardial fiber rupture were improved.(4)ELISA: the levels of IL-6,TNF-α and c Tn I in serum were significantly reduced,IL-6(Pg/ml): 16.66±5.28 vs.12.07±3.50,TNF-α(Pg/ml): 20.16±3.88 vs.10.82±2.05,c Tn I(Pg/ml): 79.01±17.25 vs.54.92±16.08,both P<0.05.The cell proliferation activity of the control group at the concentration of 0 μg/ml was 1,and the cell proliferation activity of the other groups at different concentrations of LPS was higher than that of the control group(all P < 0.001).After 24 hours of treatment with PDTC at different concentrations,the results showed that the proliferation activity of cells in the PDTC group at the concentration of 1 μmol/L was significantly lower than that in the LPS group(P < 0.001).Compared with the control group,the protein expression levels of IL-6 and TNF-α in the LPS group were significantly increased(2.015±0.365)vs(1.000±0.000),(1.961±0.380)vs(1.000±0.000)(both P < 0.05).The protein expression levels of IL-6 and TNF-α in PDTC group(1.052±0.186)and(0.717±0.401)were significantly decreased(all P < 0.05).Compared with the control group,The expression level of TLR4 protein and the ratio of p-NF-κBp65/NF-κB-p65 in TLR4/NF-κB signaling pathway in LPS group were significantly increased(1.514±0.063 vs 1.000±0.000,1.961±0.380 vs 1.000±0.000)(both P < 0.05)).Compared with the LPS group,the expression level of TLR4 protein and the ratio of pNF-κB-p65/NF-κB-p65 in the PDTC group were significantly decreased(0.882±0.240,0.717±0.401,both P < 0.05).Compared with the control group,the protein and m RNA expression levels of TGF-β1 and the m RNA expression level of type Ⅰ collagen in the LPS group were significantly increased(all P < 0.05).Compared with the LPS group,the protein and m RNA expression levels of PDTC group and the m RNA expression level of type Ⅰ collagen were significantly decreased(all P < 0.05).Conclusion PDTC can inhibit the TLR4/NF-κB signaling pathway,reduce the proliferation of LPS-stimulated rat cardiac fibroblasts and the expression of inflammatory mediators and fibrosis markers,thereby inhibiting the progression of fibrosis... |
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