| ObjectiveDiabetic macrovascular disease is eharacterized by atherosclerosis.The mechanism of atherosclerosis in diabetic macrovascular disease is un?clear,and may have close connection to oxidative stress.When diabetes happen,a oxidative stress state was generate in the body,which showed manifestations of increasing of lipid peroxidation products,decreasing of antioxidant capacity of cells and lipid levels,and abnormal glucose and lipid metabolism.Lipid peroxidation of blood vessel walls led to the generation of ox-LDL.Consuming too much ox-LDL of macrophage cell in the foaming process accompanied with a series of reactions which ultimately lead to large vascular lesions.The antioxidant is helpful for the treatment of diabetic macrovascular disease,and the main ingredients antioxidants included the total polysaccharide,total polyphenol,total protein and total saponins.In this study,we test the total polysaccharide,total polyphenols,total protein and total saponins of Lianmei granule,and preliminarily evaluate its antioxidant activity in vitro.Then we build the oxidized low density lipoprotein induced macrophage RAW264.7 foamy cell model based on the preliminary study to investigate the effect of Lianlei Granule on the foaming of RAW 264.7 macrophages induced by oxidized low density lipoprotein and its possible mechanism.Methods1.The total polysaccharide,total polyphenol,total protein,and total saponins were measured by Phonel-sulfate method,eolorimetric method using Folin reagents,coomassie brilliant blue staining method and colorimetric method using diosgenin respectively.The antioxidant activities of LM was evaluated by scavenging the 1,1-diphenyl-2-picrylhydrazyl radicals(DPPH·),super-oxide radicals(O2—·),hydroxyl radicals(·OH)and the ferrie reducing power,and the vitamin C(Vc)was used as control measure.2.We observe the degree of foaming induced by oxidized low density lipoprotein in macrophages RAW264.7 by using the method of Oil red O staining.And we explore the suitable concentration of LM for macrophage foam induced by ox-LDL by using MTT assay to detect the cell growth activity.3.We prepare different concentrations of LM granule drug-containing serum of SPF rat,and explore the effect of LM granule drug-containing serum on cells,then select the appropriate serum drug concentrations.RAW264.7 macrophages were cultured and divided into 6 groups:CDControl group(complete culture medium),?Model group(complete culture medium+80?g/mL ox-LDL),?Blank serum group(20%blank serum+80?g/mL ox-LDL),?Low dose of Lianmei granules serum group(20%Low dose of Lianmei granules serum+80?g/mL ox-LDL),?medium dose of Lianmei granules serum group dose group(20%medium dose of Lianmei drug serum+80?g/mL ox-LDL),?High dose of Lianmei granules serum group(20%high dose of Lianmei drug serum?80?g/mL ox-LDL).We observe the degree of cell foaming of ox-LDL-induced RAW264.7 cells by oil red o staining,and detect the expression of SR-B1,CD36 mRNA and protein by RT-PCR and Western Blot.Results1.The content of total polysaccharide,total polyphenol,total protein and total saponins in LM were 396.29,61.04,11.81,354.17 mg/g respectively.The scavenging effects of lianmei on the DPPH radicals,super-oxide radicals,hydroxyl radicals were obvious,and the IC50 activities were 0.16,0.59,4.36mg/mL respectively.LM also had a strong Iron reduction force.And the degree of antioxidant activity showed obvious concentration-response relationship.2.Ox-LDL MTT assay showed that the growth of RAW264.7 macrophages was inhibited with the increase of ox-LDL concentration and the time prolongation.The IC20 at 12,24,48h was 66.59,79.11 and 49.53 ?g/mL,respectively.So 80?g/mL ox-LDL at 24h should be selected to construct the cell model.At this time,the oil red 0 results were moderate and the cell lorphology were in good shape.3.The serum of the low,medium and high dose Lianmei granule of SPF rats were successfully prepared.LM granule drug-containing serum MTT experiments showed that with the increaseing in serum concentration,RAW264.7 macrophage growth gradually inhibited.The IC50 of the black serum group,low dose group,medium dose group and high dose group were 22.37%,78.61%,46.03%and 56.18%respectively.So we choose 20%as LM granule drug-containing serum concentration.4.The oil red 0 staining results showed that there were a lot of red lipid droplets in the blank serum group and the model group.There was a few red lipid droplets in the blank group.The red lipid droplets in the low,medium and high dose groups were lower than those in the lodel group,and the number of red lipid droplets decreased gradually with the increasing of the concentration of Lianmei granule containing serum.Lianmei granule containing serum high dose group of red fat droplets were significantly reduced.5.The results of RT-PCR showed that the expression of SR-B1 mRNA in Lianmei granule serum groups were significantly higher than that in blank group and model group(P<0.01);the expression of SR-B1 mRNA in the low dose group was significantly higher than that in the blank serum group;and the expression of SR-B1 mRNA in the medium group and high dose group were significantly higher than that in the blank serum group(P<0.01).The expression of CD36 mRNA in the serum group of Lianmei granule were significantly higher than that in blank group(P<0.01),however compared with the model group have a slight decline,there was significant difference between the high dose group and the model group(P<0.01),there were no significant differences between the low dose group and the medium dose group and the model group.6.The results of Western-Blot showed that the expression of SR-B1 protein in Lianmei granule serum groups were significantly higher than that in blank group(P<0.05).Among them,the high dose group was significantly higher than the blank group(P<0.01).The protein expression of SR-B1 in Lianmei granule serum group was slightly higher than that in model group,which was not statistically significant.The expression level of SR-B1 protein in the Lianmei granule serum group was significantly higher than that in the blank serum group(P<0.01),and the dose was higher than that in the blank serum group(P<0.05),the low dose group was slightly higher than the blank serum group,Which was not statistically significant.The expression of CD36 protein in low dose group was significantly higher than that in blank group(P<0.01),the expression of CD36 protein in the medium dose group was higher than that in the blank group(P<0.05),the expression of CD36 protein in the high dose group was slightly higher than that in the blank group,but there was no significant difference.The protein expression of CD36 in medium and high dose group were significantly lower than that in model group(P<0.01),and the expression of CD36 protein in low dose group was slightly lower than that in model group,and there was no significant difference.The expression of CD36 protein in the serum group was significantly lower than that in the blank serum group(P<0.01),the medium dose was lower than that in the blank serum group(P<0.05).The low dose group was slightly lower than the blank serum group,but it was not statistically significant.Conclusions1.Lianmei granule has a strong antioxidant capacity,which may provide the basis for anti-atherosclerosis.2.Ox-LDL induced RAW264.7 macrophage foam effect was significant,which can be used as a simple method to make model.3.Lianmei granule serum can inhibit ox-LDL-induced macrophage foam,which play the role of anti-atherosclerosis and may be related to the regulation of SR-B1 and CD36 expression of Lianmei granule serum. |
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