| Objective:Cerebral ischemia belongs to the category of "stroke" in traditional Chinese medicine,and is a serious disease that seriously harms human health.The prevention and treatment of cerebral ischemia is a difficult and hot topic in research.Huanglian Jiedu Decoction is a representative formula which can clear heat and remove toxicity.Huanglian Jiedu Decoction is applied to the treatment of cerebral ischemia under the guidance of the theory of "toxic damage to brain collaterals".Clinical and experimental studies show that Huanglian Jiedu decoction can play a therapeutic role in the acute and the recovery stage of cerebral ischemia,but the relevant mechanism of action needs to be further studied.Chinese herbal compound is the basic form of clinical medication.Component compatibility is one of the important forms of developing modern Chinese medicine.The purpose of this study was to explore the intervention of Huanglian Jiedu Decoction on cerebral ischemia and the relevant mechanism,and to clarify the component compatibility of Huanglian Jiedu decoction against cerebral ischemia by systematic pharmacological method,in order to provide a basis for the clinical use of Huanglian Jiedu decoction treat cerebral ischemia and provide a reference for the later investigation.Methods:1.Pharmacodynamics of Huanglian Jiedu Decoction against cerebral ischemia.To evaluate the effect of Huanglian Jiedu Decoction on cerebral ischemia,Middle cerebral artery occlusion(MCAO)Model was adopted.Male SD rats were randomly divided into sham group(Sham),model group(Model),Huanglian Jiedu Decoction high dose group(HLJDH,0.710 g/kg),Huanglian Jiedu Decoction medium dose group(HLJDM,0.355 g/kg),Huanglian Jiedu Decoction low dose group(HLJDL,0.178 g/kg),positive drug group(21.6 mg/kg,Ginaton)with the evaluation index of cerebral infarction rate and neurological deficit score.After 5 days of continuous administration,the model was constructed and the neurological deficit score and cerebral infarction rate were measured 12 h later.2.Proteomic study on the anti-cerebral ischemia effect of Huanglian Jiedu Decoction.The animals were divided into sham group(Sham),Model group and drug administration group(HLJDD,0.355 g/kg).The model was constructed after 5 days of continuous administration,and the samples were collected 12 h later for protein detection.Protein quantification,enzyme digestion,desalting and mass spectrometry were performed on the samples of each group,and the proteomic data were obtained for statistical analysis.The key targets were verified by Western blot.The animal groups and drug administration methods were the same as above.BCA method was used to determine protein concentration,electrophoresis,membrane transfer,incubation of primary and secondary antibodies,and color imaging were performed on samples from each group.3.Research on the component compatibility of Huanglian Jiedu Decoction against cerebral ischemia.The active ingredient database of Huanglian Jiedu Decoction was constructed by systematic pharmacological analysis platform(TCMSP),and the three-dimensional structures of active ingredients was obtained from PubChem database.The three dimensional structures of the callback proteins which were screened in proteomic were searched in the PDB database.The core targets and chemical components were virtual screened by Autodock to determine the component compatibility of Huanglian Jiedu decoction.The dosages of component compatibility were determined by high performance liquid chromatography and the pharmacodynamics was evaluated.The animals were divided into Huanglian Jiedu Decoction group(HLJDD,0.355 g/kg),PW1 group(berberine 17.669 mg/kg,geniposide 8.482 mg/kg,baicalin 7.913 mg/kg),PW2 group(berberine 17.669 mg/kg,geniposide 8.482 mg/kg,baicalin 7.913 mg/kg,chrysin 0.283 mg/kg),PW3 group(berberine 35.339 mg/kg,geniposide 16.964 mg/kg,baicalin 15.826 mg/kg,chrysin 0.566 mg/kg)was administered for 5 days of continuous administration,and the neurological deficit score and cerebral infarction rate were measured 12 hours later.Results:1.Pharmacodynamics of Huanglian Jiedu Decoction against cerebral ischemia.The results showed as follows:compared with Sham group,neurological deficit score in Model group were significantly increased(P<0.001)12 h after modeling,and in all drug administration groups were significantly decreased compared with Model group,HLJDM group(P<0.01),HLJDL group(P<0.05),HLJDH group(P<0.05).Compared with Sham group,the cerebral infarction rate in Model group was significantly increased(P<0.001)12 h after operation,and in all drug administration groups were significantly decreased,HLJDM group(P<0.001),HLJDL group(P<0.001)and HLJDH group(P<0.001).2.Proteomic study on the anti-cerebral ischemia effect of Huanglian Jiedu Decoction.Differential proteins were screened and 23 callback proteins were obtained,CLSTN1,CLPP and TBC1D15 were finally identified as the key targets of Huanglian Jiedu Decoction for anti-cerebral ischemia.WB results showed that compared with Sham group,the protein relative expressions of CLSTN1 and TBC1D15 in Model group were significantly up-regulated(P<0.001),while CLPP was significantly down-regulated(P<0.001).Compared with Model group,the protein relative expressions of CLSTN1 and TBC1D15 were significantly down-regulated(P<0.001),while CLPP was significantly up-regulated(P<0.001).3.Study on the component compatibility of Huanglian Jiedu Decoction against cerebral ischemia.It was determined that berberine,geniposide,baicalin,chrysin could be used as component compatibility of Huanglian Jiedu Decoction for anti-cerebral ischemia.The pharmacodynamic results of component compatibility showed that compared with Sham group,the neurological deficit score in Model group were significantly increased(P<0.001),while in each administration group were significantly decreased compared with Model group,HLJDD group(P<0.01),PW1 group(P<0.05),PW2 group(P<0.01),PW3 group(P<0.05);Compared with Sham group,the cerebral infarction rate in Model group was significantly increased(P<0.001),while in each administration group were significantly decreased compared with Model group,HLJDD(P<0.001),PW1 group(P<0.01),PW2 group(P<0.01)and PW3 group(P<0.05).Conclusion:Huanglian Jiedu Decoction can reduce the neurological deficit score and cerebral infarction rate of MCAO rats,and the medium dose of Huanglian Jiedu Decoction(0.355 g/kg)has the best effect.The improvement of Huanglian Jiedu Decoction on cerebral ischemia may be related to the regulation of CLSTN1,CLPP and TBC1D15 protein relative expressions in ischemic cortex of MCAO rats.Berberine,geniposide,baicalin and chrysin can be used as component compatibility of Huanglian Jiedu Decoction anti-cerebral ischemia,which can reduce the neurological deficit score and cerebral infarction rate of MCAO rats. |
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