Study On The Interventional Effect And Mechanism Of Wild Baicalin In Breviscapine On Atherosclerosis Of Qi Stagnation And Blood Stasis Type

Purpose:Use of rats of qi-stagnation and blood-stasis atherosclerosis to observe the effect of scutellarin on the regulation of lipid metabolism and the role of protecting the hepatic steatosis and to deeply discuss mechanism on oxidative stress as the starting point.Material and method:SPF SD male rats were randomly divided into eight groups----blank control group,H-S group,LIP5 group,XFZY20 group,SCU300 group,SCU100 group,SCU50 group and NaHS group.Rats of blank control group were fed with normal diet for 12 weeks.Rats of other groups were fed with special diet for 12 weeks,intraperitoneally injected Vim D3 for 3days（from 1st day to 3rd day）and imposed chronic and unpredictable stimulation（sound,light and electricity）for 12 weeks at the same time.At the 8th week,took 2 rats of H-S group for biochemical index test and histopathological examination,to check whether the qi-stagnation and blood-stasis model was set up.After the model was set up successfully,rats except of blank group were separately administrated LIP 5mg/kg/d,XFZY 20g（containing crude drugs）/kg/d,NaHS2.8mg/kg/d,SCU 300 mg/kg/d,100 mg/kg/d,50 mg/kg/d everyday by oral.Every week,body weight was recorded and mental status,clothing hair,and tongue demonstration of rats were scored.After 4 weeks administration,rats were fasted for 16h,and then collected blood from abdominal aorta.Biochemical index including ALT、AST、BUN、CREA、ALB、TP、LDL-C、HDL-C、CHOL、TG were tested by automatic biochemistry analyzer.FIB、PT、APTT、TT were tested by full-automatic blood coagulation analyzer.Whole blood and plasma viscosity were tested by viscosity meter.SOD activity,GSH contents,MDA concentration and H2S concentration were tested by reagent kits.Blood flow index including of tail,heart,liver,right kidney was tested by Doppler flowmeter.Weights of liver,kidney and heart were recorded and calculated index of the organs respectively.Part of the organs were stored in-80℃for further study,and the other part were for histopathological examination.Chromatographic analysis of SCU was used by HPLC.H&Einvestigated the hepatic steatosis.SOD activity,GSH contents,and MDA concentration were tested by reagent kits.The expression of HO-1,NQO1,Nrf2 and its target genes involved in antioxidant were assessed by Western blot,Quantitative Real-time PCR and immunofluorescence.The expression of PI3K P85 in PI3K/Akt signal path and active protein of phosphorylated Akt（Ser473）were assessed by Western blot and immunofluorescence.The gene expression level of Akt in liver and aorta was assessed by Quantitative Real-time PCR.The influence of generation of H2S and expression of regulatory gene CSE in qi-stagnation and blood-stasis rats’liver and aorta was assessed by Western blot and Quantitative Real-time PCR.Results:1.The effect of scutellarin on lipid metabolism and hepatic steatosis in qi-stagnation and blood-stasis ratsAfter 4-week’s administrated scutellarin,body weight of qi-stagnation and blood-stasis rats stopped decreasing and recovered gradually.The results showed that scutellarin could decrease the weight of liver,the index of liver,kidney and heart,and could protect liver（P<0.01）.The index of HDL-C,LDL-C,CHOL and AI was significantly decreased in qi-stagnation and blood-stasis rats,which showed the level of blood lipid was improved（P<0.01）.The concentration of CREA was also decreased which meant scutellarin could protect kidney.The livers of the blank group were normal in shape,size,and color,smooth in surface,and shape in edge.The livers of the model group were fulvous,rough in surface,and blunt and thick in edge and greasy,and in the surface of some livers of model group there were little yellow nodules,which showed sever fatty liver disease.The livers of scutellarin groups were better than those of model group.The histopathology research showed in model group,hepatocellular macrovesicular steatosis,structure of hepatic lobule was damaged and showed inflammatory cell infiltration,swelling,ballooning degeneration,fat vacuoles appeared in cytoplasm.All the phenomenon described above were less sever in scutellarin group.That showed scutellarin could protect the hepatic steatosis.2.The effect of scutellarin on aortsclerosis in qi-stagnation and blood-stasis ratsAfter 4-week’s administrated scutellarin,mental status,clothing hair,and tongue demonstration of qi-stagnation and blood-stasis rats were significantly improved（P<0.05）.Blood rheology index significantly decreased（P<0.05）.APTT was prolonged（P<0.05）.Blood flow index including of tail,heart,liver,right kidney was improved（P<0.01）.All those showed scutellarin could relief qistagnation and blood stasis,and the effect was equivalent to XFZY.The histopathology research showed in model group,structure of aortic wall was normal with no significant lesion（Level 0）.Lipid storage was observed in qi-stagnation and blood-stasis group with tissue necrosis and calcification in plaque and smooth muscle cells（LevelⅣ,Ⅴ,Ⅶ）.After 4-week’s administrated scutellarin,lesions were significantly improved.In SCU50group,smooth muscle cells proliferation,lipid storage,fibrous tissue proliferation calcification in plaque and smooth muscle cells（LevelⅡ,Ⅴ,Ⅶ）were observed.In SCU100 group,smooth muscle cells proliferation,lipid storage,fibrous tissue proliferation calcification in plaque and smooth muscle cells（LevelⅡ,Ⅴ,Ⅶ）were observed,but less severe than SCU100 group.In SCU300 group,smooth muscle cells proliferation（LevelⅤ）was observed.All above showed scutellarin could protect thoracic aorta from sclerosis and plaque formation.3.The effect of scutellarin on antioxidant and the mechanism in qi-stagnation and blood-stasis ratsAfter 4-week’s administrated scutellarin,the concentration of GSH of qi-stagnation and blood-stasis rats was significantly upgraded and the activity of SOD had a trend to increase（P<0.05）.For further testing index related to oxidative stress,we found that MDA level in the scutellarin groups significantly decreased（P<0.01）,and the activity of SOD and concentration of GSH significantly increased（P<0.01）.All above showed scutellarin could through regulating MDA,SOD and GSH to play the role on antioxidant.The results of Western blot and immunofluorescence showed that after 4-week’s administrated scutellarin,Nrf2 expression in liver significantly increased（P<0.01）,and antioxidant enzyme NQO1 and HO-1 expression also increased（P<0.01）.The result of Western blot also showed that Nrf2,NQO1 and HO-1 expression in thoracic aorta significantly increased,and antioxidant enzyme expression also increased.Gene expressions of Nrf2 mRNA and NQO1 mRNA in liver and thoracic aorta showed the same trend as protein expression.All above showed scutellarin could release MDA,SOD and GSH by activating Nrf2/ARE pathway to up-regulate NQO1 and HO-1.Further research of PI3K/Akt,the upper regulation signal pathway of Nrf2,showed scutellarin could significantly increase PI3K P85 and phosphorylated Akt（Ser473）protein expression in liver and thoracic aorta（P<0.01）,and p-Akt（Ser473）showed high expression in immunofluorescence test（P<0.01）.In gene test,scutellarin could significantly upgraded Akt mRNA gene expression in liver and thoracic aorta.p-Akt could promote unclear transformation of Nrf2,then regulate transcription activity of Nrf2,promoting binding of Nrf2and ARE.All above showed scutellarin could activate PI3K/Akt pathway by activating Nrf2through p-Akt.4.The effect of scutellarin on H₂S/CSE signal pathway of endogenous gas molecular in qi-stagnation and blood-stasis ratsAfter 4-week’s administrated scutellarin,the concentration of H₂S of qi-stagnation and blood-stasis AS rats significantly decreased（P<0.01）.Scutellarin could downgrade CSE expression which was the important factor for H₂S formation and regulation（P<0.01）,but gene showed high expression in liver and no significant influence of CSE mRNA in aorta.All above showed scutellarin could relate to endogenous gas molecular H₂S.Scutellarin could act on H2S/CSE signal pathway to inhibit the release of H2S,but the mechanism needed further research.Conclusion:1、Scutellarin could decrease blood lipid level,reduce hepatic steatosis,improve lipid metabolism,improve blood flow change and volume,antioxidate and protect thoracic aorta from sclerosis and plaque formation.2、This study focus on oxidative stress as the starting point,this paper discusses resistance of wild baicalin AS possible mechanism of action of qi stagnancy and blood stasis syndrome: （1）The mechanism could be enhancing the vitality of SOD,increasing the GSH content and decreasing MDA level,by activating Nrf2/ARE signaling pathway to up-regulate NQO1 and HO-1. （2）The mechanism could be raising PI3K,phosphorylating and activating Akt,and then activating Nrf2,the downstream factor of Akt.3、There was a link between qi-stagnation and blood-stasis atherosclerosis and endogenous H₂S gas signal molecules,however,its internal mechanism still needs further research;Scutellarin can act on the liver tissue of endogenous H₂S/CSE gas molecules signal pathway,regulate of its regulation on blood vessels.