The Mechanism Of HUVEC Injury By Visfatin And The Intervention Of Dingxin Recipe

Background:Atherosclerosis (AS) which is harm to human health is not only the main pathological basis of coronary heart disease (CHD), but also the most common disease of Cardiovascular disease.But the pathogenesis of AS has not yet been defined. With the Depth researching of atherosclerosis, people come to realize that inflammation is playing an important role of the development of atherosclerosis. Anti-inflammatory therapy is one of an important prevention strategy for atherosclerosis.Visfatin is a new adipokine which is a very important processing section to the occurrence and development of AS by proinflammatory. Clinical studies had shown that plasma visfatin and some inflammatory mediators such as interleukin-6(IL-6), tumor necrosis factor-a in patients with coronary heart disease increased at the same time. Other study found that plasma visfatin levels and vascular endothelial cell function of patients with type2diabete have negative correlation. It well-known that endothelial dysfunction is one of the part in the initiating procession of atherosclerosis, so protecting vascular endothelial against various inflammatory cytokines early is the key the role of curing AS.Mitogen-activated protein kinase (MAPK) is a kind of the serine/threonine protein kinase existed in eukaryotic cells. MAPK includes three major subfamilies: SAPK/JNK (stress-activated protein kinases/c-Jun NH2-termin al kinases), ERK1/2(extracellular signal-regulated protein kinase, ERK) and p38mitogen-activated protein kinase (p38MAPK). P38MAPK and JNK pathways closely related to the stress response mainly transmit inflammatory cytokines and various types of cell stress Signaling pathway. And previous studies have shown that both of P38MAPK and JNK pathways are involved in the pathogenesis of atherosclerosis. This study aimed to investigate whether the mechanism of HUVEC injury by Visfatin is related to P38MAPK and JNK signaling molecules which participate in inflammatory response involved in atherosclerosis.Dingxin Recipe (DXR) with the effect of dampness Yiqihuoxue expectorant is made up of Salvia, Panax, TPG, Melon, Poria, Codonopsis, fungus, Semen, Sophora, berberine etc. clinical Studies had shown that it has a good effect on coronary heart disease. It can aslo significantly reduced the inflammation of ischemia and reperfusion while significantly reduced the expression of JNK and P38MAPK protein and enhanced ERK protein of ischemia-reperfusion injury in rats. We want to investigate whether DXR plays a protective role in anti-atherosclerosis and protecting HUVEC, and this mechanism may be related to regulation P38MAPK and JNK pathways signaling pathways.Chapter I the effect of proliferation of HUVEC induced by visfatin and intervention of DXR serumObjective:1. The effect of different concentrations and time of visfatin on Proliferation of HUVEC2. The effect of different concentrations and time of DXR serum on Proliferation of HUVEC3. Protective effect of DXR serum in proliferation of HUVEC induced by visfatinMethod:Preparation of DXR serum:60male SD rats weighting200-250g were randomly divided into2groups,30rats in each group and were fed DXR Decoction and an equal volume of saline respectively. The dose of DXR (18.59g/kg/d) was taken twice a day for7days continuously. The30rats were treated on a water-only diet for12h before the last administration and were underwent2%sodium pentobarbital anesthesia for abdominal aortic blood sampling after the last administration for2h.Blood samples were keeped at4℃for4h, then centrifuged3500r/min for10min, at last was separated with0.22μm microporous membrane filtration sterilization, inactivated56℃for30min, aliquoted of each individual, saved at-80℃for backup.Grouping:HUVEC were divided into the following groups:Visfatin concentration gradient groups:0,50,100,200μg/L visfatin respectively stimulate HUVEC for24h.Visfatin time gradient group:100μg/L visfatin stimulate HUVEC respectively for0,6,12,24and48h.DXR serum concentrations group:0,0.25%,0.5%,1%,2%,5%,10%,20%DXR serum containing are added HUVEC for24h.DXR serum time gradient group:0.5%DXR role of serum containing respectively0,6,12,24,48h. DXR serum and Pathway inhibitor groups:P38MAPK path group:①the control group;②Visfatin+SB203580:SB203580(final concentration of10μmol/L) were pre-incubated for30min, before a final concentration of100μg/L Visfatin be added to stimulated HUVEC for24h;③Visfatin+DXR drugs serum group:5%DXR serum concentrations were pre-incubated for1h, before Visfatin(final concentration of100μg/L) be added to stimulated HUVEC for24h;④Visfatin Group:Visfatin (final concentration of100μg/L) stimulated HUVEC for24h;⑤Visfatin+blank serum:5%blank serum preincubated HUVEC for1h before Visfatin (final concentration of100μg/L) be added to stimulated HUVEC for24h.JNK pathway groups:①the control group;②visfatin+DXR drugs serum group:5%DXR serum concentrations were pre-incubated1h, before Visfatin (final concentration of100μg/L) be added to stimulated HUVEC for24h;③Visfatin+blank serum:5%blank serum preincubated HUVEC for1h before Visfatin (final concentration of100μg/L) be added to stimulated HUVEC for24h;④visfatin group:Visfatin (final concentration of100μg/L)stimulated HUVEC for24h;⑤visfatin+SP600125group:SP600125(final concentration of10μmol/L) were pre-incubated30min, before Visfatin (final concentration of100μg/L)be added to stimulated HUVEC for24h。Detection proliferation of HUVEC by MTT assay:HUVEC were seeded in ninety-six-well plates and given the appropriate treatment factors according to the experimental group. Six wells were set in each group. By MTT assay specific steps to operate their OD values measured at490nm in a microplate reader.Result:1. Effect of different times and concentrations of visfatin for proliferation of HUVECCompared with the control group, visfatin(50μg/L) can no significantly decrease the proliferation of HUVEC while visfatin (100,200μg/L) stimulated HUVEC for24h inhibit proliferation of HUVEC was significant (P<0.01). there were no significant statistically different between the two groups; at the same time, visfatin (100μg/L) had no significant inhibition of proliferation of HUVEC at6,12h. But for24,48h were significantly inhibited proliferation of HUVEC (P<0.05or P <0.01), there were no significant statistically different between the two groups.2. Different concentrations and time of DXR serum on proliferation of HUVECCompared with control group, DXR serum(0,0.25%,0.5%,1%,2%) added for24h had no significant impact proliferation of HUVEC; DXR serum(5%,10%,20%) added for24h were can significantly enhance cell proliferation (P<0.01), there were no significant statistically different between the two groups. Compared with the control group,5%DXR serum stimulated HUVEC for6,12h has no significant impact on proliferation of HUVEC;5%DXR added for24,48h were able to significantly enhanced cell proliferation (P<0.01), there were no difference statistically significant between two groups.3. DXR serum and SB203580, SP600125visfatin injury on HUVEC proliferationCompared with the control group, visfatin and the role of blank rat serum24h can be significantly inhibited HUVEC proliferation (P<0.01); compared with visfatin group,5%DXR serum, SB203580, SP600125for24h can be enhanced by the Injury visfatin HUVEC proliferation (P<0.01).ConclusionVisfatin inhibits proliferation of HUVEC, DXR serum, SB203580, SP600125can enhance the proliferation of HUVEC injury by visfatin. visfatin may be affected by p38MAPK, JNK signaling pathway for regulating inhibition of proliferation of HUVEC. The DXR serum is also possible through their regulation to protect HUVEC. Chapter II visfatin induces secretion of IL-6and TNF-a in HUVEC and the intervention of DXR serumObjective:1. The effect of different concentrations and time of Visfatin induced secretion of IL-6and TNF-a in HUVEC.2. The protective effect of DXR serum for Visfatin induced HUVEC injury.Methods and grouping:Time gradient and the concentration gradient of visfatin are the same as those in the first chapter.DXR serum and pathways related inhibitor pretreatment of cells grouped the same as those in the first chapter.The HUVEC with1×105/mL were seeded in Petri dishes, treating the cells into different groups, Cell supernatants were collected for post-processing. According to the manufacturer’s instructions.of IL-6and TNF-a kit, Measure the OD at450nm wavelength in microplate reader. The experiment was repeated more than three times.Results:1Effect of different time and concentration of Visfatin for the levels of IL-6and TNF-a in the supernatant of HUVECCompared with the control group, the group treated with visfatin(50μg/L) for24h did not significantly increased in the levels of IL-6and TNF-a in the supernatant of HUVEC, that treated with visfatin(100,200μg/L) for24h increased the levels of IL-6and TNF-a significantly (P<0.01), but the difference between the two groups was no statistically significant. Compared with the control group, those treated with visfatin(100μg/L) for6,12h IL-6and TNF-a levels did not significantly increased in the supernatants of HUVEC, those treated for24,48h could induce their expression increase (P<0.05or P<0.01), however the difference between the two groups was no statistically significant.2. The effect of DXR serum, SB203580, SP600125on the levels of IL-6and TNF-a in the supernatant of HUVECCompared with control group, expression of IL-6and TNF-a of HUVEC culture supernatant increased significantly in visfatin group and visfatin+blank serum group (P<0.01); Compared with visfatin group, DXR drugs serum, SB203580, SP600125could significantly inhibit the expression of IL-6and TNF-ainduced by visfatin.(P <0.05or P<0.01).Conclusion:Visfatin-induce increased HUVEC secretion of IL-6and TNF-a, and thus damage function of HUVEC, DXR serum, SB203580, SP600125can inhibit the secretion of IL-6and TNF-a in HUVEC induced by visfatin, and protect HUVEC function. Mechanisms may be associated with activation of the JNK pathway P38MAPK.ChapterⅢ the role of P38MAPK and JNK pathways in visfatin-induced the injury in HUVEC cells and the intervention of DXR serumObjective:1. The effect of pro-inflammatory and mechanism of visfatin were regulated by P38MAPK and JNK signaling pathway2. The weakened effect of DXR playing in proinflammatory process of visfatin was regulated by P38MAPK and JNK signaling pathways.Methods and grouping:Time gradient and the concentration gradient of visfatin are the same as those in the first chapter.DXR serum and pathways related inhibitor pretreatment of cells grouped the same as those in the first chapter. Expression of the proteins of p38MAPK and JNK pathway were detected by Western blot. HUVEC were processed as the experiments grouping, HUVEC were lysed by Western and IP cell lysate. Lysate supernatants were collected. Use BCA method for protein quantification, and then subjected the protein to western blot. With β-actin as internal control, pro-treated protein samples were separated by SDS-PAGE, followed by protein transfer to PVDF, The membrane was blocked, The membrane was probed with primary antibodies and secondary antibodies, detected by ECL Western blot detection reagents, KODAK Image Station2000MM imaging system was used for image acquisition, then measurement and analysis of the image was obtained with a gray value image processing software image Tool3.0.Results:1. Effect of different concentrations and time of visfatin on p-p38MAPK, p-JNK and total p38MAPK, total JNK protein in HUVEC.Compared with the control group, that treated with visfatin(50μg/L) for24h did not significantly increased the expression of p-p38MAPK、p-JNK in HUVEC, those treated with visfatin(100,200μg/L) for24h increased the expression of p-p38MAPK、 p-JNK significant (P<0.05or P<0.01), and the difference between the two groups was not statistically significant; the expression of p-p38MAPK, p-JNK in HUVEC did not significant increased in6,12h, under100μg/L of visfatin, while in24,48h could be significantly increased (P<0.01), the difference between the two groups was no statistically significant.2. The effect of DXR serum, SB203580, SP600125on p-p38MAPK> p-JNK and total p38MAPK, total JNK protein in HUVEC.Compared with control group, the expression of p-p38MAPK、p-JNK increased significantly in visfatin group and visfatin+blank serum group (P<0.05); Compared with visfatin group, DXR drugs serum, SB203580, SP600125could significantly inhibit the expression of p-p38MAPK、p-JNK induced by visfatin (P<0.01).Conclusion:Combining the conclusions of first two chapters shows that visfatin inhibits HUVEC proliferation and induces HUVEC increased secretion of IL-6and TNF-a through the mechanism associated with promoting p38MAPK and JNK phosphorylation. DXR serum reducing the secretion of IL-6and TNF-a in HUVEC was induced by visfatin through p38MAPK and JNK pathways, protecting HUVEC damaged by visfatin.Summary:The pro-inflammatory mechanism of visfatin was associated with the promotion of JNK, P38MAPK phosphorylation in HUVEC, indicating that DXR serum protected the HUVEC damage induced by visfatin through p38MAPK and JNK pathways.