Tumor Necrosis Factor-like Weak Inducer Of Apoptosis Promotes The Proliferation And The Expression Of Matrix Metalloproteinase9in Rat Cardiac Fibroblasts Via Nuclear Factor-κB Pathway

BACKGROUND:Myocardial fibrosis is characterized by cardiac fibroblasts accumulation and excessive deposition of collagen, it is involved in most cardiac diseases. myocardial fibrosis can increase myocardial stiffness, left ventricular systolic diastolic dysfunction, and affect cardiac electrophysiology, also lead to serious complications such as heart failure, arrhythmia and sudden cardiac death, etal. MF is a serious threat to human health. It is one of the most important pathophysiological processes involved in hypertensive heart disease (HHD). In recent years, along with the higher incidence of hypertension, the study on the prevention and cure of hypertension myocardial fibrosisi has become a international hot topic.The pathophysiology of myocardial fibrosis during hypertension is very complicated. Recently, it has been shown that inflammatory response plays an important role in the process of myocardial fibrosis. This review describes the relationships among hemodynamic stress, hormonal factors and inflammatory factors, and emphasizes the important position of inflammation during the progress of myocardial fibrosis.As a new member of the TNF superfamily of ligands, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is expressed in many tissues and cells. TWEAK can stimulate numerous cellular responses including cell proliferation, migration, proinflammatory molecule and angiogenesis production. TWEAK can by stimulating cells secrete a variety of inflammatory factor, promote myocardial into fiber cell proliferation and collagen expression increased, so as to promote the origin and development of the myocardial fibrosis, but the mechanism is not clear. Research found that nuclear transcription factors the nf-kappa B is mediated angiotensin II (angiotensin II, Ang II), TNF alpha promote myocardial fibroblasts proliferation and collagen expression the key factor of the role. Matrix metalloproteinases9(matrixmetallopeptidase9, MMP9) were confirmed in the extracellular matrix remodeling and myocardial fibrosis plays a main role in the process. But TWEAK and the nf-kappa B, MMP9myocardial fibrosis in the relationship has not been reported.OBJECTIVE:To investigate the influence and mechanism of the tumor necrosis factor-like weak inducer of apoptosis(TWEAK) act on the NF-κB pathway in cultured rat cardiac fibroblasts (CFs), and explore the the relationship between NF-κB, MMP9and and myocardial fibrosis.METHODS:1. Cultured CFs from neonatal Wistar rats were isolated by trypsinization. The CFs were observed by inverted microscope, and authenticated by waveform protein immunofluorescence. The cells of2～4generations were selected for experiment.2. qRT-PCR was used to detect the gene expression of NF-κB and MMP9. The experiment is divided into two groups--the control group:do not add Stimulating factor; TWEAK group:add rhTWEAK, making its concentration to100μg/L. Continue to cultivate cells, extracting RNA of each group to detect the expression of NF-κB after6h, and extracting RNA of each group to detect the expression of MMP9after24h.3. Western blot was used to detect the protein expression of NF-κB and MMP9.Detected the protein expression of NF-κB after intervening by TWEAK.①Concentration gradient groups:According to the end concentration of rhTWEAK, we devided groups into control group (0μg/L),1μg/L group,10μg/L group,100μg/L group and200μg/L group.②Time gradient group:Continue to culture CFs in DMEM including100μg/L rhTWEAK. Group were devided according to the incubation time:Oh group,3h group,6h group,9h group,12h group. Detected the protein expression of NF-κB after intervening by PDTC and TWEAK.①TWEAK+PDTC group:Cultivate in DMEM including100μg/L rhTWEAK and100μmol/L PDTC for6h;②TWEAK group:Cultivate in DMEM including100μg/L rhTWEAK for6h.Detected the protein expression of MMP9after intervening by TWEAK and PDTC.①TWEAK+PDTC group:Cultivate in DMEM including100μg/L rhTWEAK and100μmol/L PDTC for24h;②TWEAK group:Cultivate in DMEM including100μg/L rhTWEAK for24h;③Control group:Cultivate in DMEM for24h.4. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method.Culturing CFs to logarithmic growth period, and vaccinate CFs into96hole cell culture plate with5×103cells esch hole. After24h of culturing, changing to DMEM medium, and continuing to cultivate. After24h, we devide groups into three.①TWEAK+PDTC group:change culture solution to DMEM including100ug/L rhTWEAK and100umol/L PDTC;②the TWEAK group:change culture solution to DMEM including100ug/L rhTWEAK;③the control group:just add DMEM. Continue to culture CFs48h. Cell proliferation of each group was detcetd by MTT method.RESULTS:1.The gene expression of NF-κB and MMP9was detected by qRT-PCR. The NF-κB and MMP9mRNA expression levels of the stimulated group were higher than that of the control group.2.CFs were stimulated by different concentrations of rhTWEAK after8h, the NF-κB protein expression levels increased in a dose-dependent manner. The protein expression levels of the control group (0.510±0.011),1μg/L group (0.860±0.069),10μg/L group (1.224±0.010),100μg/L group (1.908±0.055) were gradually increased (n=3)(P<0.01). And the200ug/L group (1.942±0.069) compared the100ug/L group was not significantly increased (P>0.05)3.Stimulated by100ug/L rhTWEAK after3h, the NF-κB protein expression levels began to increase (0.815±0.063), achieve a maximum activity after6h (1.426±0.056).After the later time point(12h), the NF-κB activity obviously dcreased again (n=3)(P＜0.01)4. Stimulated by100ug/L rhTWEAK after24h, the MMP9protein expression levels of the TWEAK group (0.871±0.018) was increased significantly compared to the control group (0.232±0.010). With the100umol/L PDTC inhibiting the NF-κB pathway, the MMP9expression of the TWEAK+PDTC group (0.422±0.022) decreased significantly compared to the TWEAK group (0.422±0.022)(n=3)(P <0.01)5.The A492values of the100μg/L group were higher than that of the control group (P＜0.01),.With the100μmol/L PDTC inhibiting the NF-κB pathway, the A492values of the TWEAK+PDTC group were decreased significantly (P＜0.05).CONCLUSIONS:1.TWEAK can promote CFs proliferation and increase the NF-κB and MMP9mRNA expression in CFs. The NF-κB protein expression levels increased in a dose-dependent manner. Stimulated by100ug/L rhTWEAK, the NF-κB protein expression levels achieved a maximum activity after6h. TWEAK induced a time-dependent NF-κB translocation.2.PDTC(the NF-κB inbihitor) can significantly decrease the TWEAK induced MMP9protein expression and CFs proliferation effect, TWEAK induced myocardial fibrosis dependent on the NF-κB activation.