Study On The Intervention Of Nippostrongylus Brasiliensis And Its Derived Proteins On Occurrence And Development Of Atherosclerosis

Atherosclerosis（AS）is a chronic inflammatory disease characterized by abnormal lipid metabolism and infiltration of inflammatory cells.The inflammatory reaction runs through the whole process of its occurrence and development.Anti-inflammatory intervention has become an important direction in the prevention and treatment of atherosclerosis.Controlling or blocking the inflammatory response can effectively delay the occurrence of atherosclerosis,plaque rupture and thrombosis.It has been found that macrophages play a very important role in the process of the occurrence and development of atherosclerosis and participate in a series of pathophysiological processes such as deposition of lipid,formation of foam cell,formation and stabilization of atherosclerotic plaques.Specific inhibition of M1induction cytokines or enhancement of expression level of M2 induction cytokines can effectively alleviate the occurrence and development of AS.Epidemiological investigation shows that with the decline of worm infection rate,the incidence of allergic diseases,autoimmune diseases,metabolic diseases in the population is on the rise.The effect of worm infection or molecules of worm on the intervention of autoimmune,allergic,metabolic and other inflammation-related diseases is obvious,which provides new ideas for the prevention and treatment of such diseases.After previous research results show that hookworm infection and its source products can effectively intervene crohn’s disease,allergic asthma and host lipid metabolism by inducing the host to produce specific type II immune response,and M2 macrophage polarization.Therefore,this study proposes a scientific hypothesis on hookworm infection and its source proteins can induce the host to produce type II immune response and M2 macrophage polarization,thus interfering with the occurrence and development of atherosclerosis.In order to verify the above hypothesis,cell experiments in vitro were conducted to observe the direction of macrophages polarization induced by the proteins from Nipostrongylus brasiliensis in vitro by the index of cell morphology,mRNA,cell markers,and cytokine secretion.In addition,we used animal models of ApoE^-/-mice,in the forming process and after the formation of atherosclerosis,Nipostrongylus brasiliensis infection and their source products were used respectively to intervene the animal models,through arterial and hepatic tissue pathological slices,the level of serum lipids and cytokines detection,comprehensive evaluation of Nipostrongylus brasiliensis infection and their source products intervention effect of occurrence and development of atherosclerosis.1、Study on polarization of macrophages derived from THP-1 in vitroTo maintain the laboratory circulation of Nipostrongylus brasiliensis,collect L3and L5 worms under sterile conditions,and prepare the source protein respectively;to utilize the culture system of THP-1 cells in vitro,THP-1 cells were stimulated by PMA（500 ng/ml）,the cells were transformed into M0 cells in adherent state.LPS+IFN-γ（500 ng/ml and 100 ng/mL）,IL-4+IL-13（borh are 100 ng/mL）,L3 and L5 proteins（both are 5μg/ml）were stimulated respectively.After stimulation,M1/M2 cell morphology,M1/M2 macrophage specific gene mRNA expression,M1/M2cell markers and M1/M2 cytokine secretion were observed by microscopy,RT-qPCR,flow cytometry and ELISA,to observe the polarization direction of macrophages derived from THP-1.Results show that THP-1 was transformed into M0 cells attached to the wall by PMA stimulation,which showed the characteristic change of M1 type polarization after LPS+IFN-γstimulation,which was flat and round;after IL-4+IL-13stimulation,which showed the characteristic change of M2 type polarization,which was elongated spindle;after L3 and L5 protein stimulation,which showed the characteristic change of M2 type polarization,which was elongated spindle;The number of M1 in LPS+IFN-γstimulation group was much higher than that in the other four groups,the numbers of M2 in IL-4+IL-13 stimulation group,L3 protein stimulation group and L5 protein stimulation group were higher than that in the control group and LPS+IFN-γstimulation group.Results of RT-qPCR showed that the mRNA expression level of CCL2,TNF-α,IL-12B and other M1 macrophage related genes in LPS+IFN-γstimulation group were significantly higher than that in the other four groups,the differences were statistically significant（P<0.05）;The mRNA expression level of PPARγ,IL-10 and other M2 macrophage related genes in IL-4+IL-13 stimulation group,L3 protein stimulation group and L5 protein stimulation group was significantly higher than those in LPS+IFN-γstimulation group,the differences were statistically significant（P<0.001）;the mRNA expression level of Mrc-1 gene in IL-4+IL-13 stimulation group and L3 protein stimulation group was significantly higher than that in LPS+IFN-γstimulation group,the differences were statistically significant（P<0.001）.The results of flow cytometry showed that the number of CD86 positive cells in the negative control group and LPS+IFN-γstimulation group was higher than that in the IL-4+IL-13 stimulation group,L3 protein stimulation group and L5 protein stimulation group;the numbers of CD206 positive cells in the IL-4+IL-13 stimulation group,L3 protein stimulation group and L5 protein stimulation group were higher than that in the negative control group and LPS+IFN-γstimulation group.The results of cytokines test showed that the expression level of IL-1β,TNF-αand other M1 macrophage related cytokines in the LPS+IFN-γstimulation group was much higher compared with IL-4+IL-13stimulation group,L3 protein stimulation group and L5 protein stimulation group,the differences were statistically significant（P<0.001）;TGF-β1,IL-10 and other M2macrophage related cytokines in IL-4+IL-13 stimulation group,L3 protein stimulation group and L5 protein stimulation group were significantly higher than those in the control group and LPS+IFN-γstimulation group,the differences were statistically significant（P<0.05）.The results showed that both L3 and L5 proteins of Nipostrongylus brasiliensis could induce the M2 macrophages polarization in vitro,suggesting that the specific induction of macrophage polarization could stimulate the anti-inflammatory effect after infection with hookworm.2、Study on the intervention of Nipostrongylus brasiliensis and its products on the occurrence of atherosclerosis in ApoE^-/-miceApoE^-/-mice were fed with high fat diet for 12 weeks to establish atherosclerosis model.From the first week of modeling,the mice were injected subcutaneously with L3 protein,L5 protein and Nipostrongylus brasiliensis every 2 week to interfere with ApoE^-/-mice,and ApoE^-/-mice inoculated with adjuvant and PBS were used as control group.After 12 weeks of modeling,The intervention effect of atherosclerosis was evaluated by tissue staining and cytokine assay.The results of aortic arch section showed that L3 proteins,L5 proteins and Nipostrongylus brasiliensis had a certain intervention effect on the occurrence of atherosclerosis.After L3 protein intervention,the length of plaque,the relative content of lipid in plaque,the area of plaque and the relative area of necrotic core decreased significantly（P<0.05）;after L5 protein infection,the length and area of plaque decreased significantly（P<0.05）after N.b.infection,the length of plaque,the relative content of lipid in plaque,the area of plaque and the relative area of necrotic core decreased significantly（P<0.05）.The results of liver section showed that L3protein group and L5 protein group could improve the lipid metabolism.After the intervention of L3 protein and L5 protein,the relative content of lipid in liver was significantly reduced.The detection and analysis of cytokines showed that the Intervention groups had no significant effect on type I cytokines secretion,but they all increased the level of type II cytokines secretion to some extent.The level of IL-4secretion in L3 protein group was significantly increased from the 12th week The level of IL-4 secretion in L5 protein group was significantly increased from the 8th week,and the level of IL-10 secretion was significantly increased from the 4th week（P<0.05）;the level of IL-4 secretion in Nipostrongylus brasiliensis group was significantly increased from the 12th week（P<0.05）.The results showed that the Nipostrongylus brasiliensis and its source proteins could effectively intervene the occurrence of atherosclerotic plaques,which might be related to the type II immunity induced by Nipostrongylus brasiliensis and its source proteins.3、Study on the intervention of Nipostrongylus brasiliensis and its products on the development of atherosclerosis in ApoE^-/-miceThe atherosclerosis model was established by feeding ApoE^-/-mice with high fat diet for 20 weeks.From the 12th week of modeling,the mice were injected with L3protein,L5 protein and Nipostrongylus brasiliensis subcutaneously every other week,and ApoE^-/-mice inoculated with adjuvant and PBS were used as control group.After 20 weeks of modeling,The intervention effect of atherosclerosis was evaluated by tissue staining,serum HDL and cytokine detection.The results of aortic arch section showed that L3 protein,L5 protein and N.b.could interfere with the development of atherosclerotic plaque.After L3 protein intervention,the length and area of plaque decreased significantly（P<0.05）;after L5protein intervention,the length,area and relative area of necrotic core decreased significantly（P<0.05）;The plaque area decreased significantly after infection with Nipostrongylus brasiliensis（P<0.01）.The results of liver section showed that only L5 protein group had the effect of improving lipid metabolism,and the relative content of lipid in L5 protein group decreased significantly,but there was no significant difference between L3 protein group and Nipostrongylus brasiliensis group.Through the detection and analysis of serum HDL,the relative value of HDL of L5protein group at the 16th week was higher than that of adjuvant group,and the value of HDL increased significantly at the 20th week（P<0.05）.But there was no significant difference between L3 protein group and Nipostrongylus brasiliensis group from 12th to 20th week（P>0.05）.The analysis of cytokine detection showed that L3protein and L5 protein intervention could reduce the level of type I cytokine secretion,such as the level of IL-6 secretion significantly decreased at 20th week,the difference was statistically significant（P<0.05）;L3 protein,L5 protein and Nipostrongylus brasiliensis could up regulate some typeⅡcytokines,For example,the level of IL-10in Nipostrongylus brasiliensis group was significantly increased from the 16th week,and the level of IL-10 secretion in L3 protein group and L5 protein group was significantly increased at the 20th week（P<0.05）.The results showed that the development of atherosclerosis could be alleviated to a certain extent by the specific induction of IL-10 after intervention,which was manifested in the decrease of the size of atherosclerotic plaques and the area of necrotic core.