| Objective: Investigate the effect of SPK on the formation of AS in rats,make a prediction about the relationship between SPK and AS,and analyze the principle that lead to this result.Method:1.Randomly select 78 male SD rats,weighing 220-250 g.2.All the rats were randomly divided into 6 groups: normal group,model group,positive group,low,medium and high dose group.The requirements were as follows:A.normal group(fed with standard diet,and distilled water by gavage).B.model group(fed with high fat diet,VitD3 by intraperitoneal injection,and distilled water by gavage).C.positive group(fed with high fat diet,VitD3 by intraperitoneal injection,and 4 mg/kg of simvastatin by gavage).D.low dose group(fed with high fat diet,VitD3 by intraperitoneal injection,and 80 IU/kg of SPK by gavage).E.medium dose group(fed with high fat diet,VitD3 by intraperitoneal injection,and 160 IU/kg of SPK by gavage).F.high dose group(fed with high fat diet,VitD3 by intraperitoneal injection,and 320IU/kg of SPK by gavage).Rats in each group were fed with a regular dose of water by gavage every day.The free water drinking of rats was not limited.Measure the weight at a fixed time every 7 days,calculate the change index,and feed continuously for12 w.3.At the end of the 12 w,after anesthetizing rats with chloral hydrate,draw the required blood from abdominal aorta,remove the serum with the centrifugal method and set aside.Take off the abdominal aorta,liver,spleen,kidney and heart.Fix part of the aorta abdominalis with 4% paraformaldehyde.After freezing the other organs in the liquid nitrogen,place them in the refrigerator at-80 °.4.Measure the content of TC,TG,HDL-C and LDL-C in serum with automatic biochemical analyzer and calculate the AS index AI.Measure the content of CRP,MMP-9 in serum by Enzyme-linked immunosorbent assay(ELISA).Measure the expression of vascular endothelial growth factor(VEGF)in blood vessels by Western blotting(WB).Measure the expression of VEGF in the paraformaldehyde-fixed abdominal aorta by immunohistochemistry.Observe the pathological changes by HE staining and measure the thickness changes of the tunia intima-media with Image pro plus image processing software.Results:1.The weight changes of rats in each groupThe weight of the rats in the normal group increased steadily.The modelgroup began to grow gradually and decreased in the later stage.The positive group had no obvious fluctuation and roughly remained unchanged.The SPK low dose group increased first and then decreased.SPK medium dose group increased gradually at a fixed speed.SPK high dose group increased slightly.Generally speaking,the weight of the model group changed obviously.The weight of the normal group and the positive group did not change significantly.And the weight of the low,medium and high dose groups gradually decreased.Weight changing index shows that there were differences in the weight among groups(P <0.05).2.The changes of blood lipid levels in each groupThe growth rate of TC,TG and LDL-C was larger and and HDL-C was lower(P <0.05)in the normal group than the normal group.TC,TG,LDL-C decreased and HDL-C increased(P < 0.05)in positive group,SPK low,medium and high dose groups compared to the model group.HDL-C content in SPK low,medium and high dose groups did not change significantly compared to the normal group and positive group(P > 0.05).The calculated AS index AI of the model group was the highest(P < 0.05)compared to the normal group.AI value in the positive group and each SPK dose group decreased compared to the model group(P < 0.05).AI values of each SPK dose group had differences compared to the normal group and the positive group(P < 0.05).3.CRP inflammatory response levels of rats in each groupCRP content in serum increased in the model group compared to the normal group(P < 0.05).In terms of model group,CRP content decreased significantly in the positive group and each SPK dose group(P < 0.05).CRP content in serum had a significant difference among each SPK dose group,model group and positive group,while in terms of the normal group,there wasno big difference(P > 0.05).4.MMP-9 cell migration levels of rats in each groupIn terms of the normal group,MMP-9 content in serum increased in the model group(P < 0.05).In terms of the model group,MMP-9 content decreased significantly in the positive group and each SPK dose group(P < 0.05)and there was a very big difference.But in terms of the normal group,MMP-9 content in serum had no big difference in the positive group,low dose group(P > 0.05),while MMP-9 content in serum had very big difference in the medium and high dose group(P < 0.05).5.The cell proliferation level VEGF changes of rats in each groupMeasure the VEGF content in the abdominal aorta of rats by Western blotting.Finally obtain the relative values through measuring the absorbance values of each group,and comparing the absorbance values of the corresponding internal β-Actin.In terms of the normal group,VEGF content of vascular expression in the model group increased significantly(P < 0.05),and the expression of VEGF in both the positive group and each SPK dose group had difference with the normal group(P < 0.05).Expression of positive group had no big difference compared to the model group(P > 0.05)while had a big difference in all the SPK dose groups(P < 0.05).The expression contents were diminishing with the concentration of SPK increasing.Compare the expression contents of three SPK dose groups,we found that all of them had difference(P <0.05),and the expression contents were diminishing with the concentration of SPK increasing.6.The abdomen aorta pathological changes of rats in each groupThrough HE staining of the abdominal aorta in rats,slicing up and we found that the structure at each layer of the arterial vessels in the normal groupwas complete,the intima was smooth,and the smooth muscle cells were arranged in the normal group.The structure of the arterial vessels in the model group had varying degrees of damage: the intima drops out and defects,the media atrophies,smooth muscle cells proliferate and migrate,foam cell and lipid deposit under the intima,and some were accompanied with with the phenomenon of calcification and detachment.The positive group had no significant difference compared with normal group,while each dose group decreased under the intervention of SPK: the endothelial structure of high dose group was relatively complete,and had no significant difference compared with the normal group.The intimal was smooth in medium dose group but the medial smooth muscle cells were slightly disordered.And the intima was rough with fall-off phenomenon in the low dose group.The media was thickening significantly and protuberate towards the cavity,with a certain degree of elastic fibers degeneration,collapse and crack.Measure the thickness of intima and media in the abdominal aorta with Image pro plus software:compared with the normal group,the intima and media were significantly thicker in the model group(P <0.05);compared with the model group,there were significant differences in the thickness of intima and media among the positive group and each SPK dose group(P <0.05).The thickness of intima and media among the SPK dose groups also had significant differences(P <0.05),and the intima and media had a thinning trend with the concentration of SPK increasing.Conclusion:1.SPK have the effect of inhibition or delay on the occurrence of rat AS to a certain extent.2.SPK may inhibit or slow the formation of atherosclerosis in rats throughregulating blood lipid levels,inhibiting inflammatory response,slowing tissue cell migration and proliferative changes.3.The effect of high dose SPK on the prevention of AS is significantly better than that of medium and low dose.The ability to inhibit the inflammatory response is better than simvastatin,and the effect of slowing the migration and proliferation of tissue cells is better than simvastatin.However,its effect on depressing the blood-fat is no better than simvastatin. |
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