Protective Effect Of Laminarin L01 On Vascular Endothelial Cells In Chronic Inflammation Rats And Lipopolysaccharide-induced Injury Of Brain Microvascular Endothelial Cells

Objective: A rat model of LPS chronic inflammation was established and the LPS stimulation model of HBMECs was cultured in vitro.To investigate the protective effect of Laminaria L01 against LPS inflammation on vascular endothelial cells and related mechanisms,to develop new ideas for the prevention and treatment of cardiovascular and cerebrovascular diseases.Methods:(1)Establishment of chronic inflammatory rat model:LPS(0.4 mg/kg)was injected via tail vein once a week for 4 weeks.The inflammatory response was evaluated by WBC count and hypersensitive C-reactive protein content in serum.(2)Animal experiment:SD rats were randomly divided into 6 groups.In addition to the blank control group only injected with sterile saline,the other 5 groups received intraperitoneal injection of dexamethasone(10 mg/kg)and L01 high,medium and low dose(50,30,10mg/kg)on the day after the first injection of LPS.LPS group was injected with the same volume of aseptic saline once a day for 4 weeks.24 hours after the last administration,the number of whole WBC was couted,the hs-CRP in serum was measured by ELISA,the expression of e NOS,i NOS and COX-2 m RNA were detected by RT-PCR and the related products were examined by density and half-quantitative analysis after electrophoresis.(3)Cell experiments:HBMECs were cultured in vitro and randomly divided into blank control group,LPS group,L01-H control group,LPS+L01-L group,LPS+L01-M group,and LPS+L01-H group.Several doses of L01 groups were pretreated with the corresponding dose of L01 for 1h,and then added with 40μg/m L of LPS for48 h.Detection of NO Level in HBMEC supernatant by Griess method;Quantitative Real-time PCR was used to detect the expression of IL-6m RNA,e NOS m RNA and i NOS m RNA in cells;Western blot technique was used to detect the protein levels of T-e NOS and T-i NOS in the cells,as well as the phosphorylation level of P-e NOSSer1177.Results:(1)LPS increased WBC counts and serum hs-CRP levels in rats,down-regulated the expression of e NOS m RNA and up-regulated the expression of i NOS m RNA and COX-2 m RNA.These changes can be antagonized after 4 weeks of therapeutic administration of different concentrations of L01.(2)LPS increased NO level in HBMEC culture medium,down-regulated the expression of e NOS m RNA and protein,and down-regulated the phosphorylation of P-e NOS ser1177,up-regulated theexpression of i NOS m RNA and protein.But different concentrations of Laminarin L01 can reversed these changes.Conclusion: L01 can reduce the release of inflammatory factor IL-6induced by LPS,inhibit the increase of whole blood leukocyte and hs-CRP,and reduce the excessive NO expression in cell culture fluid.Up-regulated the expression of e NOS gene and protein,and promoted the phosphorylation of P-e NOSSer1177,down-regulated the expression of i NOS gene and protein.The results suggest that Laminaria L01 can resist endothelial cell damage caused by LPS,thus maintaining the basic stability of the cardiovascular and cerebrovascular environment.