| Objective(1)To investigate the correlation between myocardial fibrosis and AMPK/Sirt1/NF-κB signaling pathway mediated inflammatory response in the aging process of rats.(2)To study the amelioration of total saponins of Panax japonicas(TSPJ)on anti-myocardial fibrosis and its mechanism through AMPK/Sirt1/NF-κB signaling pathway in aging rats.Those studies provided theoretical basis and experimental support of the clinical development of TSPJ for the prevention and treatment of myocardial fibrosis in aging rats.Methods(1)Male Sprague-Dawley rats were divided into 3-month-old(3M)group,9-month-old(9M)group,15-month-old(15M)group and 24-month-old(24M)group according to age,each group of 10 rats.(2)30 male SD rats aged 18 months old were randomly divided into 3 groups(aging model group,low-dose TSPJ group and high-dose TSPJ group),each group of 10 rats.TSPJ groups were given TSPJ at different doses(10and 60 mg/kg/d,p.o.)consecutively for 6 months,thus aging model group was treated with equal volume of saline for 6 months until 24 months old.Another 10 rats aged 6 month(6M)were used as young-controlled group.After the last dose,taking the weight of the rats and injecting the anesthetic into the abdominal cavity,then killing the rats.The main detection indicators were as follows,1)The changes of myocardial morphological in each group were observed by haematoxylin-eosin(HE)staining.2)Masson staining observed the changes of collagen deposition in rat hearts.3)Immunofluorescence were used to detect the protein expression changes and localization of fibrocyte marker vimentin and NF-κBp65.4)The changes in the protein expressions of Vimentin,p-AMPK,Sirt1,Ac-NF-κBp65,IL-1β,TNF-α were detected by Western Blot.5)RT-PCR was used to detect the marker of myofibroblast α-SMA,collagen-related protein COL1α2,COL3α1,inflammatory correlation factor IL-1β,TNF-α,IL-6,MCP-1 and matrix metalloproteinase MMP2,MMP9 m RNA expression levels.Results(1)1)HE staining showed that the myocardial fibers in 3M and 9M were orderly,with clear structure and compact structure.The myocardial fibers were disordered in 15 M,and the muscle fibers were broken,and the gap was enlarged and accompanied by inflammatory cell infiltration.The muscle fibers of 24 M were obviously disturbed,and the myocardial clearance increased significantly,accompanied by a large number of inflammatory cells infiltrating.2)Masson staining results showed that collagen was mainly deposited in the extercellular matrix and surrounding blood vessels,and the collagen deposition increased with age,with the most prominent among the 24 M rats.3)Immunofluorescence showed that Vimentin was located on the edge of the cytoplasmic membrane of the cytoplasm,and it was distributed in thick and thin green lines or filaments,and interwoven into a network structure.With the increase of age,Vimentin expression gradually increased,and the two sides of the cell gradually extended into a long and narrow spindle,among which Vimentin was the strongest expression in the cell elongation in 24 M.Western Blot also showed that the protein expression of Vimentin in the heart of 24 M rats was significantly increased compared with those of 3M and 9M rats.4)Immunofluorescence results showed that there were less expression of NF-κBp65 in the hearts of 3M,9M and 15 M rats,while the expression of NF-κBp65 in 24 M rats was significantly increased,and was mainly concentrated in nucleus.5)Western Blot results showed that Ac-NF-κBp65 protein in the heart of 24 M rats increased significantly compared with that of 3M,9M and 15 M rats,while the Sirt1 protein level was significantly decreased in the heart of 24 M rats compared with 3M,9M and 15 M rats.At the same time,the expression level of p-AMPK in the heart of 15 M and 24 M rats were significantly reduced compared with that of 3 months old rats.6)RT-PCR results showed that the m RNA expression levels of TNF-α,IL-1β and IL-6,MCP-1 in the heart of 24 M rats were significantly increased compared with those of 3 and 9 months of age.The m RNA expression of IL-6 and MCP-1 in the heart of 24 M were also significantly increased compared with that of 15 M rats.(2)1)Low and high dose of TSPJ can attenuate myocardial disorder,decrease inflammatory infiltration,and reduce collagen deposition in aging rats.2)Low and high dose TSPJ can effectively promote the up-regulated expression of p-AMPK and Sirt1 protein in senescence myocardium,while reduce the acetylation level of NF-κBp65 protein.3)TSPJ can significantly inhibit the inflammatory factors expression of TNF-α and IL-1β compared with aging model group.4)Compared with aging model group rats,the m RNA expression levels of myofibroblast markers α-SMA,collagen-related protein COL1α2,COL3α1 and matrix metalloproteinase MMP2,MMP9 were down-regulated in TSPJ treated group.Conclusion(1)Myocardial fibrosis is aggravated during aging,and its mechanism is related to decrease the protein expression of p-AMPK and Sirt1,promotes the acetylation of NF-κBp65 protein,which will induce myocardial inflammatory response.(2)TSPJ can promote the expression protein of p-AMPK and Sirt1,and inhibit the acetylation of NF-κBp65 protein in the heart tissue of aging rats,thereby reducing myocardial inflammatory response and improving the myocardial fibrosis with aging. |
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