| IntroductionAtherosclerosis(AS) is a complex process of multi-factor and multi-stage vascular inflammatory disease, involved activation of endothelial cells, extracellular matrix degradation, infiltration of inflammatory cells, smooth muscle cell hyperplasia and change a serious number of cytokines and chemokines. The current researches on anti-atherosclerosis mainly focus on reducing lipid, anti-inflammatory, antioxidant, and so on. Statins is praised highly clinical medicine, play an important role in improving AS and reduced complication. However, some cases confirmed the existence of statins has a side effect. Looking for new treatment for the treatment of AS is fitting for the urgent demands. Now have more experimental evidence in support of extractions of housefly larvae in anti-atherosclerosis, our previous research has separated the lower molecular weight polypeptide of the extractions of housefly larvae(E, <30KD) and also found its effective for improving AS in reducing lipid, antioxidant and anti-inflammation. Based on described above, this study intends to understand the function and the mechanism of the lower molecular weight polypeptide of the extractions of housefly larvae(E) anti-AS. lipopolysaccharide(LPS) was used for building cell inflammation model of endothelial cells(EC), smooth muscle cells(SMC), macrophages(Mφ) firstly, then was constructed to blank control(control), negative control(model), Simvastains(Sim), 5 or 20 or 80 μg/ml E(E5, E20, E80) groups and interacted with the two cells or incubated individual. Cell proliferation, migration, secretion were examination by PI labeling, wound heal assay and ELISA assay.Objective1. To explore the effect of housefly larvae extractions on LPS-induced EC individual culture and interaction SMC or Mφ;2. To explore the effect of extractions of housefly larvae on LPS-induced SMC individual culture and interaction EC or Mφ;3. To explore the effect of extractions of housefly larvae on LPS-induced Mφ individual culture and interaction Mφ or SMC.Methods1. The effect of extractions of housefly larvae on LPS-induced EC individual culture and interaction SMC or Mφ: Primitive culture techniques used to obtaining vascular endothelial cells from male rat, passages 4 ~ 8 were taken to observe the expression of CD31 by immune fluorescence and flow cytometry respectively, small tube formation on the extracellular matrix gel were also used for cell identification. 100 ng/ml lipopolysaccharide(LPS) was used to incubation with EC for 12 hours to built inflammation model, then the LPS-induced cell were used for single culture, co-culture and constructed to blank control(control), negative control(model), Simvastains(Sim), 5 or 20 or 80μg/ml E(E5, E20, E80) groups for 12 hours instead. TNF-α, IL-6 and VEGF were examination by ELISA, cell migration was tested by Boyden chamber assay and wound heal assay, cell proliferation were check by PI labeling and MTT, respectively. In cell interaction, LPS-induced EC were feed into the upper chamber, SMC or Mφ were seeded into the lower chamber. Cell interaction was used pore size 0.4 μm transwell plate and cell migration was used pore size 8 μm. Cell proliferation, migration, secretion were examination according to the described above.2. The effect of extractions of housefly larvae on LPS-induced SMC individual culture and interaction EC or Mφ: Primitive culture techniques used to obtaining vascular smooth muscle cells from male rat, passages 4 ~ 8 were taken to observe the expression of alpha-SM by immune fluorescence, western blotting and flow cytometry respectively. Cellsingle culture or co-culture and cell proliferation, migration, secretion were examination according to 1.3. The effect of extractions of housefly larvae on LPS-induced Mφ individual culture and interaction Mφ or SMC: Primitive culture techniques used to obtaining bone marrow cells from male rat, these cells were suspended containing 50 ng/ml colony-stimulating factor for 7 days for bone marrow-derived Mφ cultures. After all, cells were taken to observe the expression of CD11b/c by immune fluorescence and flow cytometry respectively, staining with Switzerland-Giemsa and ink intake were also used for cell identification. Cell single culture or co-culture and cell proliferation, migration, secretion were examination according to 1.4. Statistical analysisAll datas were expressed as the mean±standard error of the mean( sx±). All statistical analyses were performed by SPSS(version 13.0 for Windows) statistical software. Quantitative variables were analyzed by one-way analysis of variance. When the data are homogeneity, multiple comparison was used LSD method. If the data are not homogeneity, multiple comparison was used Dunnett T 3 method. The difference was statistically significant when P<0.05.Result1. The effect of extractions of housefly larvae on LPS-induced EC individual culture and interaction with SMC or Mφ: The cells became more closely packed, exhibiting a cobblestone-like appearance about two weeks. When the cells reached confluence, the standard passage procedures were used for subculture. Interestingly, small tube formation on the extracellular matrix gel and its passages changed in shape gradually from oval to polygonal but the cellular surface expression of the endothelial marker CD31 by immunofluorescence and flow cytometry, suggesting that the cells were epithelia. MTT results showed that the strong promotion effect of LPS on the proliferation of EC and after LPS-induced would increase EC in G2/M phase concomitant with a decrease in G0/G1 and S phase, so did increasing the rates of migration. Besides, the concentrations of TNF-α,IL-6, VEGF between blank control and model groups were significantly different(P<0.05), implied LPS-induced EC inflammation. However, Simvastains and different concentration of extractions of housefly larvae can hardly inhibitory the proliferation and migration of EC, degenerated the concentrations of inflammation though decreased the ratio of G2/M phase and migrate distance, reduced the concentrations of TNF-α, IL-6, VEGF(P < 0.01). When EC interacted with SMC, E5 and E80 can inhibitory the proliferation and migration of EC, degenerated the concentrations of inflammation to some extent(P<0.01), except simvastains(P>0.05). When EC interacted with Mφ, Simvastains and different concentration of extractions of housefly larvae can hardly inhibitory the proliferation and migration of EC, degenerated the concentrations of inflammation though decreased the ratio of G2/M phase and migrate distance, reduced the concentrations of TNF-α, IL-6, VEGF(P<0.01).2. The effect of extractions of housefly larvae on LPS-induced SMC individual culture and interaction with EC or Mφ: About two weeks later, the smooth muscle cells appeared narrow, elongated, and ribbon shaped. Smooth muscle cells usually banded together to form a meshwork of growth. As the growth became confluent, Stellate-like patterns were formed. And cytoplasm were abundance of α-SM by immunofluorescence, western blotting and flow cytometry. The results showed that the strong promotion effect of LPS on the proliferation of SMC and after LPS-induced would increase SMC in G2/M phase concomitant with a decrease in G0/G1 and S phase, so did increasing the rates of migration. Besides, the concentrations of TNF-α, IL-6, VEGF between blank control and model groups were significantly different(P<0.05), implied LPS-induced SMC inflammation. However, E20 and E80 can inhibitory the proliferation and migration of SMC, degenerated the concentrations of inflammation to some extent(P<0.01), simvastains showed no effect on LPS-induced SMC(P>0.05). When SMC interacted with EC, Simvastains and different concentration of extractions of housefly larvae can hardly inhibitory the proliferation and migration of SMC, degenerated the concentrations of inflammation though decreased the ratio of G2/M phase and migrate distance, reduced the concentrations of TNF-α, IL-6, VEGF(P<0.01). When SMC interacted with Mφ, E5 can inhibitory the proliferation and migration of SMC, degenerated the concentrations of inflammation to some extent(P<0.01), simvastains showed no effect on LPS-induced SMC(P>0.05).3. The effect of extractions of housefly larvae on LPS-induced Mφ individual culture and interaction with EC or SMC: Bone marrow cells were subsequently maintained in media containing M-CSF after the initiation of they separated. One week later, the cells, a progressive increase in volume of the cytoplasm and serosal macrophage containing dense bodies, increased pseudopodium and growth attached. Cellular surface marker by anti-CD11b/c body tested by immunofluorescence and flow cytometry, cell were showed purple when staining with Switzerland-Giemsa and black after ink intake. The results showed that the strong promotion effect of LPS on the proliferation of Mφ and after LPS-induced would increase Mφ in G2/M phase concomitant with a decrease in G0/G1 and S phase, so did increasing the rates of migration. Besides, the concentrations of TNF-α, IL-6, VEGF between blank control and model groups were significantly different(P<0.05), implied LPS-induced Mφ inflammation. However, Simvastains and different concentration of extractions of housefly larvae can hardly inhibitory the proliferation and migration of Mφ, degenerated the concentrations of inflammation though decreased the ratio of G2/M phase and migrate distance, reduced the concentrations of TNF-α, IL-6, VEGF(P<0.01). When Mφ interacted with EC, E5 can promote cell proliferation but inhibit cell migration of Mφ, no effect on the concentrations of inflammation. E20 and E80 can inhibit Mφ proliferation and migration, degeneration the concentrations of inflammation to some extent(P<0.01), simvastains showed no effect on LPS-induced SMC(P>0.05). When Mφ interacted with SMC, E80 can promote cell proliferation but inhibit cell migration of Mφ, no effect on the concentrations of inflammation. simvastains showed no effect on LPS-induced Mφ when Mφ interacted with SMC(P>0.05).ConclusionAs result showed above, the concentrations of TNF-α, IL-6, VEGF and the ratio of G2/M phase and migrate distance were increasing after LPS-induced. In vitro assay confirmed that the extractions of housefly larvae would can significantly inhibit cell proliferation, migration and inducing cell cycle arrest after LPS-induced, especially EC, Mφ, EC whencocultured with Mφ, SMC when interacted with EC. In sum, the extractions of housefly larvae showed a potential effect on anti-AS through anti-inflammation, degenerated cell migration and inhibited cell proliferation. |
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