The Inhibitory Effect Of Rosiglitazone On Porphyromonas Gingivalis Infection-accelerated Atherosclerosis In Apo E^-/-mice

Obiective:Porphyromonas gingivalis(P.gingivalis) is an important pathogen of chronic periodontitis. Studies confirm that P. gingivalis periodontal inoculation or blood injections could accentuate inflammation of the vascular wall, and accelerate the progress of Atherosclerosis(AS). The anti-inflammatory treatment is considered to be feasible treatment strategy of inhibiting the progression of AS, because AS is thought to be a chronic inflammatory disease.Peroxisome Proliferator-activated receptor-γ(PPAR-γ) belongs to the nuclear hormone receptor superfamily and is a ligand-activated nuclear transcription factor, which plays an important role in lipid and glucose metabolism. At present, it is found to have extensive inhibition of inflammation. Rosiglitazone(RSG) is a synthetic PPAR-γ specific agonist. P.gingivalis and its products have pathogen-associated molecular patterns(PAMPs), which could activate the host defense system of Toll-like receptors(TLRs), leading to activation of downstream signaling pathways such as nuclear factor-κB( NF-κB). This research intends to further study the mechanism by which P.gingivalis infection accelerates AS, and explore PPAR-γ activator of rosiglitazone in P.gingivalis TLRs- NF-κB signaling pathway.Experiment Ⅰ : The inhibitory effect of rosiglitazone in P.gingivalis-challenged human aortic endothelial cells(HAECs)Methods: HAECs were cultured in vitro, and were challenged according to the following groups respectively : control groups, RSG groups, P.gingivalis groups, P.gingivalis +RSG groups. Gene expressions of monocyte chemotactic protein 1(MCP-1)、interleukin 1β(IL-1β)、TLR-2、TLR-4、PPAR-γ and intercellular adhesionmolecule-1(ICAM-1) in HAECs were identified by real time quantitative PCR(RT-q PCR). MCP-1 and IL-1β in the culture medium were determined by ELISA. TLR-2、TLR-4、PPAR-γ and ICAM-1 in HAECS were analyzed by Western Blot. The activation of NF-κB was determined by electrophoretic mobility shift assay(EMSA).Results: P.gingivalis increased the expression of MCP-1、IL-1β、ICAM-1、TLR-2 and TLR-4 in concentration-dependent manners(MOI: 0、25:1、100:1 和 200:1) and promoted the activation of NF-κB in HAECs. RSG increased the expression of PPAR-γ in concentration-dependent manners(0、2.5、5 和 10μM)in HAECs. RSG inhibited the expression of MCP-1 、 IL-1β 、 ICAM-1 、 TLR-2 and TLR-4 in concentration-dependent manners, and inhibited the activation of NF-κB in P.gingivalis-challenged HAECs.Conclusion: P.gingivalis induces inflammatory response in HAECs and leads its injury via TLRs/NF-κB signaling pathway, which is inhibited by RSG.Experiment Ⅱ : The inhibitory effect of rosiglitazone on Porphyromonas gingivalis bacteremia-accelerated atherosclerosis in Apo E-/- MiceMethods: At 10 weeks of age the Apo E-/- mice were randomly divided into four groups: control groups, RSG groups, P.gingivalis groups, P.gingivalis +RSG groups. Mice were injected via caudal vein with sterile PBS or live P. gingivalis and injected intraperitoneally with saline or RSG(10 mg/kg) respectively. With weekly infections of a total of 10 times, the animals were euthanized at 24-h after the last infection. Tumor necrosis factor(TNF-α)、MCP-1 and IL-1β in the serum were determined by ELISA. The levels of serum lipid were determined by standard enzymatic test kits. The atherosclerotic plaque area was determined by en face quantification with aortic tree stained by sudan Ⅳ. The expressions of PPAR-γ、ICAM-1、TNF-α、MCP-1、TLR-2 and TLR-4 in aortic tissues were detected by Immunohistochemistory staining and RT-q PCR. Protein expressions of TLR-2 and TLR-4 in aortic tissues were analyzed by Western Blot. The activation of NF-κB in aortic tissues was determinedby EMSA.Results: RSG treatment activated the expression of PPARγ in the aorta, reduced the P.gingivalis bacteremia accelerated atherosclerotic plaque areas, reduced serum TNF-α、MCP-1 and IL-1β levels and total cholesterol(Tc) and low density lipoprotein cholesterol(LDL-c), decreased the expression of ICAM-1、TNF-α、MCP-1、TLR-2 and TLR-4 in aortic tissues caused by P.gingivalis bacteremia, inhibited activation of NF-κB in aortic tissues advanced by P.gingivalis bacteremia.Conclusion: P.gingivalis bacteremia accelerates atherosclerosis by inducing local and systemic inflammatory response via TLRs/NF-κB signaling pathway in Apo E-/-mice, which is inhibited by RSG.Experiment Ⅲ : The inhibitory effect of rosiglitazone on Porphyromonas gingivalis periodontal infection-accelerated atherosclerosis in Apo E-/- miceMethods: At 10 weeks of age the ApoE-/- mice were randomly divided into four groups, control groups: intraperitoneal injection of saline; P.gingivalis groups: periodontal ligation and inoculation of P. gingivalis, intraperitoneal injection of saline; P.gingivalis +RSG groups: periodontal ligation and inoculation of P. gingivalis, intraperitoneal injection of RSG(10 mg/kg); RSG groups: intraperitoneal injection of RSG(10 mg/kg). Experimental animals were challenged once a week. After 10 weeks, the animals were euthanized and the specimens were collected. Periodontal destruction was analyzed by methylene blue staining.The atherosclerotic plaque area was determined by en face quantification with aortic tree stained by sudan Ⅳ. TNF-α、MCP-1 and IL-1β in the serum were determined by ELISA. The levels of serum lipid were were determined by standard enzymatic test kits. The expressions of PPAR-γ、ICAM-1、TNF-α、MCP-1、TLR-2 and TLR-4 in aortic tissue were detected by Immunohistochemistory staining and RT-q PCR. Protein expressions of TLR-2 and TLR-4 in aortic tissues were analyzed by Western Blot. The activation of NF-κB in aortic tissues was determined by EMSA.Results: Periodontal infection of P.gingivalis caused significantly periodontal destruction. Periodontal infection of P.gingivalis increased the aortic atherosclerotic plaque areas in Apo E-/- mice. RSG treatment activated the expression of PPARγ in the aorta, reduced the P.gingivalis periodontal infection accelerated atherosclerotic plaque areas, reduced serum proinflammatory cytokines levels and Tc and LDL-c, decreased the expression of ICAM-1、TNF-α、MCP-1、TLR-2 and TLR-4 in aortic tissues caused by P.gingivalis periodontal infection, inhibited activation of NF-κB in aortic tissues advanced by P.gingivalis periodontal infection.Conclusion: Periodontal infection of P.gingivalis accelerates atherosclerosis by inducing local and systemic inflammatory response via TLRs/NF-κB signaling pathway in Apo E-/- mice, which is inhibited by RSG.