Study On The Mechanism Of Shenmayizhi Decoction On Vascular Cognitive Impairment Rats Based On Mitochondrial Associated Protein Signal Pathway

Vascular cognitive impairment(VCI)includes a series of cognitive impairment caused by vascular factors,covering all types of cognitive impairment related to vascular diseases.At present,due to the heterogeneity of clinical manifestations and the limitations of current diagnostic criteria,the epidemiology of VCI is difficult to carry out.With the increasing aging of the population and the rising incidence of cerebrovascular diseases,the incidence and prevalence of VCI were also increased.The etiology and pathological mechanism of VCI are complex,which is related to inflammatory reaction,free radical damage,cholinergic damage,apoptosis and heredity closely.However,VCI is still considered to be preventable.At present,the main method to treat VCI is to treat the vascular disease and other VCI risk factors,such as hypertension and diabetes.Considering there is no effective drug to improve the disease,our research group drew up a compound prescription of traditional Chinese medicine ShenmaYizhi decoction for the prevention and treatment of VCI,and become the hospital preparation of Xiyuan Hospital of China Academy of Chinese Medical Sciences.The decoction is composed of Panax ginseng C.A.Mey(Renshen),Gastrodia elata Bl.(Tianma),Euonymus alatus Sieb.(Thunb.)(Guijianyu),and Ligusticum chuanxiong Hort.(Chuanxiong),which has the effect of replenishing qi and increasing intelligence,promoting blood circulation and removing blood stasis.Previous animal experiments and clinical studies have shown that Shenma Yizhi decoction can improve the cognitive and neurological functions of VCI rats and patients.In this study,Shenma Yizhi decoction was used as an intervention drug to detect its effects to improve the ability of learning and memory and the ultrastructure of mitochondria in brain tissue of VCI model rats,as well as its effect on AMPK/PPAR?/PGC-1?/UCP2 signal pathway,and to further explore the mechanism of its action on brain energy metabolism,so as to provide scientific basis for its prevention and treatment of VCI.ObjectiveTo determine the extraction technology and to clarify the effective chemical composition of ShenmaYizhi decoction.To observe the effects of ShenmaYizhi decoction on the ability of learning and memory,serum inflammatory factors and oxidative stress related indexes in rats with VCI caused by chronic cerebral ischemia induced by bilateral common carotid artery ligation.To explore the pathological mechanism of brain energy metabolism and mitochondrial disorders related proteins AMPK,PGC-1?,PPARa and UCP2 in rats with CCH.Meanwhile,to detect the intervention mechanism and possible target of ShenmaYizhi decoction on cerebral energy metabolism disorder induced by CCH.MethodsThe main chemical constituents of ShenmaYizhi decoction were analyzed systematically by liquid chromatography-mass spectrometry(LC-MS).70 SD rats,SPF grade,half male and half female.The model of chronic cerebral ischemia was established by ligation of bilateral common carotid arteries.After successful operation,the VCI rats were divided into four groups of 10 rats each randomly:The model group was administered an equal volume of distilled water.The donepezil group was administered 0.45 mg/kg/d donepezil,which is equivalent to the clinical dosage.The SMYZ-H group was administered 11.88 g/kg/d SMYZ,which is 4 times higher than the clinically equivalent dosage.The SMYZ-L group was administered 2.97 g/kg/d SMYZ,which is the clinically equivalent dosage.A sham-operated group was used as the control group and administered an equal volume of distilled water.The rats in the 4 groups were treated by gavage with equal volumes of liquid and the indicated concentration of drug diluted in distilled water for 8 consecutive weeks.8 weeks later,learning and memory abilities of rats were measured by Morris water maze,the pathomorphological changes of hippocampal CA1 region were observed by HE staining,the contents of Ach and acetylcholinesterase AchE in serum were measured by colorimetry,and the contents of serum inflammatory factors IL-1? and TNF-? were determined by enzyme linked immunosorbent assay(Elisa).The contents of glutathione GSH,MDA,SOD and GSH-PX were measured by colorimetry and non-enzymatic immunoassay.Mitochondria were extracted from brain tissue for further detection.The changes of the number of mitochondria were observed by immunofluorescence,and the ultrastructure and morphology of mitochondria were observed by transmission electron microscope.The effects of ShenmaYizhi decoction on the expression of AMPK,PPAR?,PGC-1? and UCP2,ATP5A mRNA were observed by RT-PCR,and the the level of AMPK,pAMPK,PPAR?,PGC-1? and UCP2,ATP5A proteins were observed by Western Blot.Results1 The composition of ShenmaYizhi decoction is stable and clearCombined with literature reports,the main chemical constituents of ShenmaYizhi decoction were analyzed systematically by LC-MS,many main chemical constituents such as ginsenoside,ferulic acid,protocatechuic acid,gastrodin and ?-sitosterol in ShenmaYizhi decoction were determined.2 Effect of ShenmaYizhi decoction on learning and memory ability of vascular cognitive impairment rats2.1 The effect of ShenmaYizhi decoction on spatial learning and memory ability of rats with VCINavigation trials:compared with the first day,the EL of all rats on the second day of training was significantly decreased(P<0.05),suggesting that all rats showed certain learning and memory ability.After repeated measurement and analysis of variance,compared with the control group,the EL of the model group was significantly prolonged from the third day,and the difference was statistically significant(P<0.05).It was suggested that the spatial learning ability of the model group was significantly lower than that of the control group.Compared with the model group,the EL of SMYZ-H group and Donepezil group were significantly shortened,and the difference was statistically significant.It is suggested that both ShenmaYizhi decoction and Donepezil hydrochloride can improve the spatial learning ability of rats with VCI.Probe trials:Compared with the sham group,the number of times crossing the original platform,the stay time in the target quadrant and the active distance in the target quadrant were significantly reduced in the model group,and there was significant difference between the two groups(P<0.01).Compared with the model group,the times of crossing the original platform in Donepezil group and SMYZ-H group were increased significantly,and the staying time and moving distance in the target quadrant were increased significantly in the SMYZ-H group,and there was significant difference between the two groups(P<0.05,P<0.01).2.2 The effect of ShenmaYizhi decoction on the pathological morphology of hippocampal CA1 region of rats with VCIAnder light microscope,the hippocampal CA1 region of sham group showed that the neurons in hippocampal CA1 region were arranged tightly and neatly,the intracellular structure was complete,the cell membrane was clear,the cytoplasm was rich,the nuclear membrane was obvious,the nucleolus was obvious,and no neuronal degeneration or necrosis was found.In the model group,the neurons in the hippocampal CA1 region were scattered and sparse,the number of cells decreased significantly,the number of layers decreased,some of them showed nuclear pyknosis,and the phenomenon of cell vacuole degeneration and even necrosis could be seen.Compared with the model group,the arrangement of neurons in hippocampal CA1 region of Donepezil group and SMYZ-L and SMYZ-H groups were improved to a certain extent,the number of cells was increased significantly,the cell structure and membrane was more complete,and there was occasional vacuolar degeneration and few cell necrosis.2.3.The effect of ShenmaYizhi decoction on the contents of AchE and Ach in the brain tissue of rats with VCICompared with the sham group,the content of Ach in the model group decreased significantly,while the content of AchE increased significantly,and there was significant difference between the two groups(P<0.01).Compared with the model group,the content of Ach in Donepezil Hydrochloride group and SNYZ-H group increased significantly(P<0.05),while the AchE content decreased significantly in Donepezil Hydrochloride group and SMYZ-H group(P<0.05).There was no significant difference among the treatment groups(P>0.05).2.4 The effect of ShenmaYizhi decoction on the contents of serum inflammatory factors IL-1? and TNF-? in rats with VCICompared with the sham group,the contents of IL-1? and TNF-? in the model group were higher than those in the sham group significantly(P<0.01).Compared with the model group,the content of IL-1? and TNF-? in Donepezil hydrochloride group and SMYZ-L,SMYZ-H groups decreased significantly(P<0.05,P<0.01).2.5 The effect of ShenmaYizhi decoction on the contents of GSH,MDA,SOD and GSH-PX in serum of rats with VCICompared with the sham group,the contents of GSH and GSH-PX in the model group decreased significantly,while the content of MDA increased significantly,and the difference was statistically significant(P<0.05,P<0.01).Compared with the model group,the contents of GSH and GSH-PX in Donepezil Hydrochloride group and SMYZ-H group were increased significantly,while the MDA content was decreased significantly(P<0.05),and the SOD content in SMYZ-H group was higher than that in the model group significantly(P<0.05).There was no significant difference among the treatment groups(P>0.05).3 The effect of ShenmaYizhi decoction on brain mitochondria of rats with VCI3.1 The effect of ShenmaYizhi decoction on the number of mitochondria in brain tissue of rats with VCIAfter the intervention of ShenmaYizhi decoction,the fluorescence showed that the number of mitochondria increased to a certain extent,indicating that ShenmaYizhi decoction could increase the number of mitochondria in VCI rats caused by bilateral common carotid artery ligation.3.2 The effect of ShenmaYizhi decoction on the ultrastructure of mitochondria in the brain of rats with VCIThe structure of mitochondria in the sham group was normal basically,neatly arranged,the membrane morphology was complete basically,the mitochondrial crest was dense,and there was no obvious swelling and vacuole formation.In the model group,the structure of mitochondria in brain tissue changed obviously,the mitochondrial membrane was blurred,part of the membrane was ruptured,the mitochondrial crest was broken,loose and dissolved,accompanied by the decrease or disappearance of matrix granules.The structure of mitochondria was significantly improved in Donepezil hydrochloride group and SMYZ-H and SMYZ-L group,the mitochondrial membrane was clear and the mitochondrial crest increased.It is suggested that ShenmaYizhi decoction can improve the mitochondrial structure of brain tissue of VCI rats.3.3 The effect of ShenmaYizhi decoction on the expression of AMPK,PPAR?,PGC-1?,UCP2 and ATP5A mRNA in the brain of rats with VCICompared with the sham group,the expression levels of AMPK,PPAR?,PGC-1?and ATP5A mRNA in the model group were lower than those in the control group significantly(P<0.05,P<0.01).After the intervention of ShenmaYizhi decoction,the expressions of AMPK,PPAR?,PGC-1? and ATP5A mRNA were higher than those in the model group significantly(P<0.059,P<0.01).The expression of UCP2 mRNA in the model group was higher than that in the sham group,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of UCP2 mRNA in Donepezil Hydrochloride group,SMYZ-H and SMYZ-L group decreased significantly(P<0.05).It is suggested that ShenmaYizhi decoction can promote the energy metabolism of mitochondria in VCI rats through AMPK/PPAR?/PGC-1?/UCP2 pathways.3.4 The effect of ShenmaYizhi decoction on the expression of AMPK,PPAR?,PGC-1?,UCP2,ATP5A in the brain of rats with VCICompared with sham group,the protein expression levels of pAMPK,PPAR?,PGC-1?,UCP2 and ATP5A in the model group were lower than those in the model group significantly(P<0.05,P<0.01).Compared with the model group,the protein expression levels of pAMPK,PPAR?,PGC-1?,UCP2 and ATP5A in the SMYZ-H group were higher than those in the model group significantly(P<0.05,P<0.01).But there was no significant difference in AMPK protein expression,suggesting that AMPK may participate in this pathway through phosphorylation.Conclusion1 The composition of ShenmaYizhi decoction is clear,which provides a chemical basis for the study of pharmacological action and mechanism of effective prevention and treatment of VCI.2 The structural changes of brain mitochondria and the disturbance of energy metabolism may be the important pathological basis of VCI.3 ShenmaYizhi decoction can improve the learning and memory ability of VCI rats induced by chronic cerebral hypoperfusion,improve the pathological morphology of hippocampal tissue,protect neurons,enhance the function of cholinergic system,inhibit inflammatory reaction and improve the ability of antioxidation.Its mechanism is related to increasing the expression of mitochondrial key proteins such as AMPK,PPAR?,PGC-1?,UCP2 and improving energy metabolism in brain tissue.