The Mechanism Of Leech On Anti-atherosclerosis And Regulating Serum Lipids

ObjectiveAtherosclerosis(AS)is a chronic progressive inflammatory disease,the occurrence of the disease involves a variety of pathogenesis.The activation,migration and proliferation of vascular smooth muscle cells(VSMCs)are the important pathological processes in the occurrence of AS and studies have pointed out that the imbalance of proliferation and apoptosis of VSMCs is an important part of plaque formation during the development of AS,and hyperlipidemia is a risk factor for the development of AS.Previous studies have confirmed that Chinese medicine leech has a certain inhibitory effect on the proliferation and phenotypic transformation of VSMCs in early AS rats,and it can also reduce serum lipids,but the exact mechanism is not yet known.p38 MAPK signal transduction pathway is an important pathway that can regulate cell proliferation and death,and can participate in many physiological processes such as cell proliferation,differentiation and apoptosis;ACAT-2,Fas,and HMGCR are the related enzymes involved in the regulation of lipid metabolism and synthesis in the liver..This study was to observe the effect of Chinese medicine leech on early-stage AS rats in order to explore the role of p38 MAPK signal transduction pathway in regulating the proliferation and apoptosis of rat VSMCs and the dynamic changes of serum lipids during the development of AS,exploring the mechanism of Chinese medicine leech in anti-atherosclerosis and reducing serum lipids in AS rats.Methods1.Animal models were established: High fat diet(HFD)feeding combined with intraperitoneal injection of vitamin D3 were taken to establish the early AS model.Forty healthy male Wistar rats were divided into 4 groups using a digital randomized table method: normal control group,model group,low and high leech(Chinese Herbal water fleas dose 0.6,1.2 g·kg-1,respectively)dosage group.The rats in the low-dose and high-dose leech were given a one-time intraperitoneal injection of vitamin D3 injection 600,000 IU·kg-1 and fed with HFD at the same time;the rats in the normal control group were given intraperitoneal injections of normal saline while normal diet was given.2.Intervention with traditional Chinese medicine leech: The prepared Chinese medicine leech liquid was administered to the two water phlegm intervention group according to the dose(10m L·kg-1),and the distilled water was given to the normal control group and the model group,giving to gavage once a day for 8 weeks.Rats were weighed once a week to adjust the amount of gavage.3.Collection and detection of specimen : At the beginning of the experiment and at the end of the fourth week,blood was collected from the tail vein of each group to detect blood lipid levels;at the end of the experiment(at the end of the 8th week),all rats were sacrificed,and rat serum,aorta,and liver were taken.The liver index of rats was calculated based on body weight;the levels of TG,TC,LDL-C,and HDL-C in rat serum were measured by a biochemical analyzer;the TGF-β1 levels was determined by enzyme linked immunosorbent assay(ELISA);Immunohistochemical Histological Chemitry(IHC)was taken to detect the expression of PCNA and Caspase-3 in VSMCs.TUNEL assay was used to detect the apoptosis of rat VSMCs.Western blot was used to detect the expression of MKK3,P38 and C-myc proteins in VSMCs,and the expression levels of ACAT-2,Fas and HMGCR in liver tissue.The rat aortic arch hematoxylin-imine was observed the pathological changes under light microscope after red(HE)staining;Hepatic tissue was observed the iver lipid deposition after oil red O staining.Results1.The changes of body weight and liver index in rats: There was no significant difference in body weight in all groups when starting this study(P> 0.05).With the process of the experiment,the body weight of the rats in the normal control group showed a steady growth trend,the body weight of the rats in the model group increased slowly and the weights was less than the normal control group(P <0.01).The body weight of the rats in the groups intervented by leech showed a steady growth and was significantly higher than the model group(P <0.01),and it had no obvious difference between the two leech intervention groups(P <0.01).In the model group,the weight of liver and liver bioindex were higher than the normal group(P<0.01),and those in the leech intervention group were lower than the model group,and the difference was statistically significant(P <0.01).2.The changes of serum lipids: It had no obvious difference in the level of serum TG,TC,LDL-C and HDL-C in the rats in all groups when starting experiment(P>0.05).At the 4th and 8th week,the levels of TC and LDL-C in model group were significantly higher,and the HDL-C level was lower than the normal group(P <0.01).Compared with the model control group,the levels of TC and LDL-C in leech inervention group were significantly lower and the HDL-C level was higher(P <0.01),but there was no significant difference between the high dose group and the low dose group(P> 0.05).3.The changes of serum TGF-β1: The level of TGF-β1 in the model group elevated,and the difference was statistically obvious,when comparing with the normal group(P < 0.01).The serum TGF-β1 in the two leech intervention groups were lower than the model group(P < 0.01),and it had no obvios difference between the two leech intervention groups(P > 0.05).4.The changes of MKK3,P38 and C-myc in VSMCs of rats: The levels of P38 and C-myc in the model group was higher than the normal group(P <0.01),while there was no significant difference in MKK3(P >0.05).Compared with the model group,the MKK3,P38 and C-myc decreased in leech intervention groups(P <0.01).Meanwhile,compared with the leech low dose group,the level of MKK3 and C-my elevated slightly in the leech high dose group(P <0.05),and there was no significant difference in MKK3 in two groups(P >0.05).5.The changes of ACAT-2,Fas and HMGCR in the liver of rats: Compared with the normal control group,the levels of ACAT2,Fas and HMGCR in the liver of the model group elevated obviously(P <0.01).Compared with the model group,the ACAT-2,Fas and HMGCR in leech intervention rats decreased(P <0.01).Meanwhile,the level of HMGCR in the high dose group was lower than that in the low dose group(P <0.05),and there was no significant difference in ACAT-2 and Fas levels between the two groups(P >0.05).6.The changes of PCNA,Caspase-3 in VSMCs of rats: The level of PCNA in VSMCs of the model group was obviously higher than the normal group(P <0.01),while Caspase-3 elevated slightly(P >0.05).Compared with the model group,the PCNA decreased and Caspase-3 elevated in leech intervention groups(P <0.01).Meanwhile,compared with the leech low dose group,the level of PCNA increased and Caspase-3decreased in the leech high dose group,but there was no significant difference(P >0.05).7.The comparison of the Apoptosis Rate of Rat VSMCs: It had no significant difference between the normal and model group in the percentage of apoptosis rate of the VSMCs(P >0.05).Compared with the model control group,the percentage of apoptosis rate of VSMCs in rats of each intervention group was significantly higher(P <0.01),indicating that the apoptosis rate of VSMCs in rats treated with leech intervention group was higher than that of the model control group.8.The analysis of correlation between TGF-β1 and PCNA,Caspase-3,MKK3,P38,C-myc,ACAT-2,Fas,HMGCR and apoptosis rate of VSMCs: It had a negative relation between TGF-β1 in serum and Caspase-3,apoptosis rate of VSMCs(P <0.05)and the expression level of TGF-β1 in serum was positively correlated with the expression levels of MKK3(P <0.05),PCNA,P38,C-myc,ACAT-2,Fas and HMGCR(P <0.01).9.The morphological changes of aortic arch: It showed that Aortic structure of each layer kept normal,complete,smooth,and VSMCs arranged neatly in normal control group,without significant cholesterol crystal deposition and plaque formation after HE staining.And in model group,aortic arch showed intimal thickening,visible plaque which protruded into the cavity,VSMCs proliferation,and arranged in disorder with obvious cholesterol crystal deposition and plaque formation.Compared with the model group,leech intervention groups had different degrees of ease in the morphological changes.In the low dose of the leech group,the aortic thickening of the aorta was mild,and a small amount of plaque was formed in the aorta and there was a small amount of cholesterol crystal deposition in the plaque,and the VSMCs were arranged neatly.The high dose of the leech group showed a small amount of plaque and cholesterol crystal deposition,VSMCs arranged more neat,fewer plaques.10.Visual observation of rat liver and oil red O staining: In the normal control group,the rat liver had a smooth surface,a ruddy color,a sharp edge,a reddish-brown texture,and a relatively tough texture;in the model group,the liver was swollen and swollen,with a smooth surface and blunt edges,thick,pale,with a soft,grainy texture,accompanied by greasy sensation;Compared with the model group,the liver of the low-dose and high-dose leech group has a pale reddish-brown color,a slightly blunt edge,and a soft texture,the volume is slightly smaller,and the graininess and slightly greasy.There was no obvious deposition of lipid droplets in the liver of normal control rats.There was obvious lipid deposition in the liver of rats in the model group,which was diffuse.Compared with the model group,the deposition of lipid droplets in the liver of rats with high and low doses of leech decreased,but the high dose group of leech The deposition of lipid droplets in rat liver was significantly lower than that in the low-dose group.ConclusionsThe results of the study indicate that the leech can interfere with the development of AS and the possible mechanism may be as follows:1.Leech can reduce the levels of TC,LDL-C in early stage rats,increase HDL-C expression level,then reduce lipid deposition in blood vessel intimal of rats.2.Leech can reduce the liver index of rats and affect the expression of ACAT-2,Fas and HMGCR in lipid metabolism in rat liver to reduce serum lipids.3.Leech can reduce the expression of serum TGF-β1 in rats to reduce the proliferation of VSMCs.4.Leech can reduce the expression of PCNA to inhibit the proliferation of VSMCs,up-regulate the expression of Caspase-3 to promote the apoptosis of VSMCs.5.Leech can regulate the expression levels of MKK3,P38 and C-myc proteins in the p38 MAPK signaling pathway regulating the proliferation and apoptosis of VSMCs to affect the process of AS.