Influence Of Baicalin On Hyperlipemia And NF-κB Signaling Pathways Induced By Lipopolysaccharide

ObjectiveInflammation is a vascular system of living tissue damage factor defense responses of what happened in many diseases, inflammation of the pathological process plays an important role. Atherosclerosis is one of the main diseases that harm human health. AS is acknowledged to be a chronic inflammatory response characteristics of the pathological process, its development is always associated with inflammation, and chronic inflammation mediated its occurrence and development process. AS is a disease of heart head blood-vessel pathological basis. Researches on the pathogenesis of AS are always hot spot. In order to lower the mortality and disability of cardiocerebrosvascular disease, it is very important to study the reasons, mechanism, treatment and prevention of AS. In recent years, relationship of Gram-negative bacterium cell walls important component-lipopolysaccharide (LPS) and AS become research focus. Many inflammatory cells and cell gene participate in the process of AS, especially as great inflammatory cell in Atherosclerosis plaques, macrophage cell can Produce variety of inflammation of the media and the inflammation factor by the NF-κB way to Promote the formation and development of Atherosclerosis plaques. Our topic group in the study confirmed the Baicalin of resistance as effect, but for the role of the mechanism is not entirely clear. This study aims to explore:(1)The regulation role of baicalin to signal transduction of NF-κB. (2) Baicalin intervention hyperlipidemia mice lipid disorders.MethodsThe present study includes two parts:In vivo experiments and vitro experiments. 1. In vivo experiments:Establishing of C57BL/6 mice hyperlipidemia animal model and intervention role of baicalin. It mainly includes these experiment:(1) Hyperlipemia C57BL/6 mice animal models were established with LPS intraperitoneal injection and high-fat diet.(2) Animals were given baicalin treatment for 7 weeks but not any food for 12 hours, then we pick eye to collect blood and separate serum. Adopt enzymatic assay animal blood lipid levels, Including total cholesterol (TC), triglycerides(TG), high-density lipoprotein cholesterol(HDL), evaluate baicalin in serum lipid content for prevention and control of influence.2. In vitro experiments:Explore regulation role of baicalin to signal transduction of NF-κB, the experiments include:(1) Mice macrophage (RAW264.7 cells) were cultured in vitro(2) Effect of Baicalin on expression of TNF-α,IL-6 in LPS-stimulated macrophage cells.(3) Effect of Baicalin on expression of TNF-α,IL-6 mRNA in LPS-stimulated macrophage cells.(4) Effect of Baicalin on expression of Nuclear NF-κB p65 protein in LPS-stimulated macrophage cells.Results1.Lipid levels of each laboratory mice(1) Establishment of hyperlipemia model miceUse C57BL/6 mice built hyperlipidemia animal model with LPS intraperitoneal injection and high-fat die. Compared to the blank group, simple high-fat model group serum TC, TG content increased (P<0.001, P<0.01), there is statistical significance. High-fat+LPS model group serum TC, TG content increased significantly. Compared to the blank group, there is statistical significance (P<0.001). Simple high-fat group HDL-C content become lower than the blank group. And Fatty+LPS model group HDL-C content obviously reduce, compared with the blank group (P<0.05). Tip mouse model of hyperlipemia founded.(2) Baicalin effect on serum lipid level of Hyperlipemia mice Compared to model group, HDL-C levels of both baicalin high-dose and low-doses groups have been increased. Among them baicalin in low-dose rise especially clear (P<0.001), and the high dose of baicalin group (P<0.01), both of them are statistically significant.2. Mice macrophage (RAW264.7 cells) were cultured in vitro The normal RAW264.7 cells has the typical "cobblestone" style, and is round or irregular shape.3.Effect of Baicalin on expression of TNF-αand IL-6 in LPS-stimulated macrophage cells.Normal macrophages has little expression of TNF-αand IL-6, Baicalin in 100μmol/L concentration has no significant effect, compared with the normal control group there is not statistical significance. LPS in 1μg/mL concentration can significantly stimulate Expression of TNF-αand IL-6 in RAW264.7 cells. Compared with the normal control group (P<0.01). In 10-100μmol/L concentration range, baicalin pretreatment are effective in suppressing the expression of TNF-a and IL-6 in LPS-stimulated macrophage cells. Compared with LPS group, there is significant difference (P<0.01).4. Effect of Baicalin on expression of inflammatory cytokines in LPS-stimulated macrophage cells.Expression of TNF-αand IL-6 gene in normal macrophages is extremely low, the expression is not affected if adding baicalin alone. The expression of TNF-αand IL-6 were markedly up-regulated in macrophage cells stimulated with LPS. Pretreated with baicalin inhibited LPS-induced TNF-αand IL-6 expression in mRNA levels. Compared with the LPS group, baicalin in 10μmol/L, 50μmol/L and 100μmol/L concentration could inhibite expression of TNF-αand IL-6 (P<0.05,0.01). It shows baicalin can effectively restrain expression of TNF-αand IL-6 gene in macrophage cells stimulated by LPS.5.Detect Nuclear NF-κB p65 protein by immune imprintingNF-κB exists in cytoplasm in an inactie three polymer form in normal macrophage.If it suffer exocellular stimulate activation into the nucleus, NF-κB could cause downstream gene expression.The NF-κB pathway is the key to make inflammatory factor express that induced by LPS. Western Blot results show that normal macrophages nuclei has little expression of NF-κB p65, and the expression of NF-κB p65 was significantly increased stimulated with LPS. Pretreated with baicalin inhibited LPS-induced NF-κB p65 expression in protein levels.ConclusionsBaicalin intervention rise HDL-C level of Hyperlipidemia mice. High-density lipoprotein have fight AS role. It confirmed that Baicalin reduce atherosclerosis risk factors through the lipid role. This, to a certain extent suppressed AS occurs.LPS can activate signal molecules of NF-κB in macrophages, and increase expression of nuclei NF-κBp65 protein. The expression of TNF-αand IL-6 in LPS-stimulated macrophage cells were markedly up-regulated. Baicalin pretreatment reduce inflammation caused by LPS, reduce macrophages NF-κB p65 into nuclear and the activity of NF-κB, thus it could limit expression of inflammation factors TNF-αand IL-6. Baicalin might effectively down regulated TNF-αand IL-6 expression in LPS-induced macrophage cells by inhibited the activation of NF-κβ. It could be speculated that amelioration of atherosclerosis damage by baicalin might contribute, in part at least, to the suppression of inflammation.This research, the method of in vivo and in vitro experiments, discusses baicalin in lipid, signaling pathways leading to the activation and inflammatory factor of several aspects of inhibiting AS role and mechanisms.