Effect Of Oriental Latrine Fly Larvina-effective Fraction On Mouse Model Of Atherosclerosis And Lymphocytes In Vitro

Atherosclerosis (AS) was a kind of disease endangering the human health seriously, Itwas considered cause by imbalance of immune regulation. Oriental Latrine Fly Larvinawas known as the maggots, also called the grain insects. It is a traditional Chinesemedicinal material, included in the fourth volume of the Compendium of Materia Medicainsect Ministry. It has the functions of clearing detoxication, resolving food stagnancy andenhancing the immunity. In recent years, the development and utilization of OrientalLatrine Fly Larvina in foods, nutritious foods and medicine aroused extensive concern insociety. According to separating by Sephadex, screening anti-inflammatory activity in vitro,freeze-drying, we found the active site which molecular weight below30KD has a betteranti-AS effect both in vivo and in vitro，and its mechanism was related to immuneregulation. As mentioned above, immune reaction is the key factor to the process of AS.Lymphocytes are the main cells of immune system, and lymphocytes and their subsetscooperate and contradict each other in immune response, thus maintained the homeostasisof immune system. So this paper focused on the influence of Oriental Latrine FlyLarvina-Effective Fraction on proliferation, differentiation and secretory function oflymphocytes in the animals and in vitro, combined with anti-atherosclerosis effect analysis.Study on effect and mechanism of the Oriental Latrine Fly Larvina-Effective Fraction inanti-AS from the perspective of immune regulation. Objective:According to detecting the serum lipid、inflammation factor and immune molecule，observing the change of the histomorphology of spleen, the change of the ratio betweenCD4+and CD8+to observed the anti-AS effect of Oriental Latrine Fly Larvina-EffectiveFraction (OLFL-EF）， cleared and definited the role of OLFL-EF on AS lesions,differentiation and secretory function of lymphocytes in vivo. Observed the influence ofOLFL-EF on proliferation, differentiation and secretory function of the splenic lymphocyte,T and B cells in vitro, further clearly the effect and mechanism of the OLFL-EF onlymphocyte. Study the effect and mechanism of OLFL-EF on AS from the perspective ofimmune regulation both in vivo and vitro.Methods:1、Studying on the anti-AS effect of OLFL-EF in mice AS model.(1) Established AS mouse model. Combined with LPS intramuscular injection and highcholesterol diet to built AS model, all mice randomly divided into four groups: normalgroup, negative group, Simvastatin group (25mg/kg/d) and OLFE-EF group(200mg/kg/d). After treatment for6weeks, it could observed the therapeutic effect.(2) The effect of OLFL-EF on serum lipids. Total cholesterol (TC) was determined bycholesterol oxidase, Triglycerides (TG) was determined by triglycerides phosphateoxidase, Low-density lipoprotein (LDL) was determined by polyethylene sulfateprecipitation method, high density lipoprotein (HDL) was determined by catalyticprecipitation method.(3) The effect of OLFL-EF on inflammation factors and immune molecular. Theconcentration of the TNFα and IL-6and IL-10and TGF-β1were determined byELISA.(4) The effect of OLFL-EF on thoracic aorta and the spleen.①Observed the pathologicalchange of mice thoracic aorta by HE staining, and detected thoracic aorta intima areaand media area, calculated the ratios of intima/media area.②Observed thepathological change of mice spleen by HE staining.③Detected the ratios of CD4+ and CD8+in spleen by immunofluorescence.2、Research on the effect of OLFL-EF on lymphocytes in vitro.(1) Detected the effect of OLFL-EF on splenocyte.①Primary culture mouse splenic lymphocyte，observed proliferative effect ofdifferent concentration of OLFE-EF on splenocyte which has stimulated by ConA or LPSby MTT assay，and defined safe concentration of OLFL-EF.②The effect of OLFL-EF on splenocyte stimulated by ConA. The splenocyte wererandomly divided into three groups: normal group, ConA group and ConA/OLFL-EF group.The splenocyte has stimulated by ConA，then detected proliferative effect of OLFL-EF atdifferent time points by MTT assay, the cell cycle distribution, the change of the ratio ofCD4+and CD8+T cells、the ratio of Treg cells was detected by flow cytometry.③The effect of OLFL-EF on splenocyte stimulated by LPS. The splenocyte wererandomly divided into three groups: normal group,LPS group and LPS/OLFL-EF group.The splenocyte has stimulated by LPS，then detected proliferative effect of OLFL-EF atdifferent time points by MTT assay, the cell cycle distribution was detected by flowcytometry.(2) The effect of OLFL-EF on T cells. To isolate T and B cells by using the nylon woolcolumn. T cells were randomly divided into three groups: normal group, ConA groupand ConA/OLFL-EF group. To detecte the proliferation effect of OLFL-EF on T cellsat different time points by MTT assay. The concentration of TNFα, IL-6, and IL-10andTGF-β1were determined by ELISA.(3) The effect of OLFL-EF on B cells. Three groups were assigned: normal group, LPSgroup and LPS/OLFL-EF group. To detecte the proliferation effect of OLFL-EF on Tcells at different time points by MTT assay.Results:1、Studying on the anti-AS effect of OLFL-EF in mice AS model.(1) Established AS mouse model. Combined with LPS intramuscular injection and highcholesterol diet could successfully built AS model. The thoracic aorta of model micerevealed characterized atherosclerotic pathological changes: intimal thicking, endothelial cells unevenly and necrosis, smooth muscle cells of media was disarranged,foam cell infiltration. The thoracic aorta intima area and media area in the model groupincreased obviously(P＜0.01), and the ratios of intima/media area increased as well(P＜0.01). In the model group,the level of TNFα and IL-6were increased obviously(P＜0.01), and IL-10and TGF-β1were significantly decreased(P＜0.01).(2) The effect of OLFL-EF on serum lipids. After6weeks therapy of OLFE-EF, the levelof TC (negative group vs. OLFE-EF group:6.39±0.14vs.4.53±0.05), TG (negativegroup vs. OLFL-EF group:3.42±0.04vs.2.24±0.12) and LDL (negative group vs.OLFL-EFgroup:3.37±0.07vs.1.91±0.08) were decreased highly (P＜0.01), the levelof HDL (negative group vs. OLFL-EF group:0.83±0.15vs.1.05±0.07) were increasedhighly (P＜0.01).(3) The effect of the inflammation factors and immune molecular: The level of TNFα(negative group vs. OLFL-EF group:1.74±0.03vs.1.24±0.14) and IL-6(negativegroup vs. OLFL-EF group:74.36±0.53vs.41.62±0.03) were decreased significantly(P＜0.01), the level of IL-10(negative group vs. OLFL-EF group:25.23±0.31vs.59.89±0.21) and TGF-β1（negative group vs. OLFL-EF group:82.27±0.11vs.187.14±0.18）were increased highly (P＜0.01).(4) The effect of OLFL-EF on thoracic aorta and the spleen.①The pathological change of mice thoracic aorta. after6weeks therapy of OLFE-EF,mice thoracic aorta was signigicantly thinner, the edge of intima and media was clearlydistinguish, and the accumulation of foamy macrophages reduced. The thoracic aortaintima area (negative group vs.OLFL-EF group:8551.9±188.45vs.1874.2±132.32),media area(negative group vs.OLFL-EF group:17215±243.52vs.14385±241.19)increased apparently(P＜0.01), and the ratios of intima/media area (negative groupvs.OLFL-EF group:0.497±0.134vs.0.130±0.027) increased(P＜0.01).②Observed the pathological change of mice spleen. After6weeks therapy of OLFE-EF,the edge of acini lienalis is clearly distinguish, B cells are generated in the germinalcenter, the edge of periarterial lymphatic sheath is smaller, the number of T cells wasdecreased.③The change of ratio of CD4+and CD8+in spleen. After6weeks therapy of OLFE-EF, accumulation of T cells was reduced, and the ratio of CD4+and CD8+was decreased.2、Research on the effect of OLFL-EF on proliferation, differentiation and secretoryfunction of lymphocyte in vitro.(1) Detected the effect of OLFL-EF on splenocyte.①Primary culture of splenic lymphocyte. The splenic lymphocyte is grown insuspension, roud, referactive index, and uniformly dispersed in the culture medium.②Determine safe and effective experimental concentration. A range of5-100μg/mLOLFL-EF could could promote proliferation of splenic lymphocyte significantly (P＜0.01). This promotion presented concentration-dependent effects. OLFL-EF could highlypromote cells proliferation in a concentration of40μg/mL. While the concentrationhigher the40μg/mL, the proliferation effect was decreased (which may be cytotoxic). So,the experimental select40μg/mL for the best safe and effective concentration.③The effect of OLFL-EF on splenocyte induced by ConA.With a range of12h-72h, OLFL-EF could significantly increased splenocyte inducedby ConA(P＜0.01). This enhance effect presented a time-dependent model with a range of12h-48h. After48h (ConAgroup vs. ConA/OLFL-EF group:0.407±0.003vs.0.698±0.004)treatment, the rate of proliferation (ConA group vs. ConA/OLFL-EF group:91.67±0.29vs.180.11±0.47) was the highest.Flow cytometry experiment reveal that OLFL-EF could reduce the percentage of cellswhich in G0/G1phase(P＜0.01) while rising the percentage of cells in S phase and G2/Mphase (P＜0.01). After OLFL-EF processing for48h, its S-Phase fraction (SPF)(12.23±0.2%) and Proliferation index (PI)(21.50±0.96%) reached the maximum value.The changes of CD4+/CD8+T cells ratio in splenocyte. Treated with OLFL-EF for24h、48h and72h, the ratio of CD4+was reduced(P＜0.01). The ratio of CD4+/CD8+wasreduced at24h (ConA group vs. ConA/OLFL-EF group:1.60±0.35vs.1.34±0.89)、48h(ConA group vs. ConA/OLFL-EF group:1.64±0.21VS1.26±0.28)、72h(ConA groupvs. ConA/OLFL-EF group:1.63±0.43vs.1.24±0.31)(P＜0.01).The ratio of Treg cells was increased significantly after24h (ConA group vs.ConA/OLFL-EF group:6.21±0.13vs.7.49±0.22),48h(ConA group vs. ConA/OLFL-EF group:6.96±0.32vs.10.22±0.78)(P＜0.01).④The effect of OLFL-EF on splenocyte induced by LPS:With a range of12h-72h, OLFL-EF could significantly increased splenocyte whichhas stimulated by LPS (P＜0.01). The enhance effect presented time-dependent at a rangeof12h-48h. After48h (LPS group vs. LPS/OLFL-EF group:0.356±0.003vs.0.565±0.002) treatment, the rate of proliferation (LPS group vs. LPS/OLFL-EF group:71.37±0.93vs.102.15±0.32) was the highest.Flow cytometry test cell cycle distribution reveal that OLFL-EF could reduced thepercentage of cells which in G0/G1phase(P＜0.01) while rising the percentage of cells in Sphase and G2/M phase (P＜0.01). After OLFL-EF processing for72h, the SPF value(6.03±0.59%) and the PI value(13.24±0.45%) are the largest. OLFL-EF promoted cells enterinto the S and G2/M phase from G0/G1phase in72h.(2) The effect of OLFL-EF on T cells.OLFL-EF could significantly recreased proliferation of T cells (P＜0.01), aftertreatmented24h (ConA group vs. ConA/OLFL-EF group:0.305±0.003vs.0.265±0.01)and48h (ConA group vs. ConA/OLFL-EF group:0.315±0.032vs.0.242±0.008). Theratio of proliferation of T cells at48h (ConAgroup vs. ConA/OLFL-EF group:5.15±0.13vs.-7.41±0.06) was lowest. OLFL-EF could inhibited the proliferation of B cells T cells at48h.The level of TNFα (control group vs. OLFL-EF group:1.22±0.13vs.1.14±0.145)and IL-6(control group vs. OLFL-EF group:40.58±0.26vs.33.42±0.13) were decreasedsignificantly (P＜0.01), the level of IL-10(control group vs. OLFL-EF group:63.17±0.45vs.72.47±0.18) and TGF-β1（control group vs. OLFL-EF group:82.27±0.11vs.190.31±0.23vs.199.14±0.48）were increased highly (P＜0.01).(3) The effect of OLFL-EF on B cells:OLFL-EF could significantly increased B cells (P＜0.01), after treatmented24h(LPS group vs. LPS/OLFL-EF group:0.323±0.004vs.0.345±0.021) and48h (LPSgroup vs. LPS/OLFL-EF group:0.346±0.017vs.363±0.013). The ratio of proliferationof B cells at48h (LPS group vs. LPS/OLFL-EF group:27.48±0.13%vs.33.62±0.09%). OLFL-EF could promote the proliferation of B cells at48h.Conclusions:OLFL-EF has certain anti-AS effect. OLFL-EF can be effectively regulatedproliferation, differentiation and secretory function of lymphocytes in AS, in order tomaintain the immune homeostasis, which may be one of the mechanisms of anti-AS.