| ObjectiveThe aim of this study was to explore the action mode of effective ingredients in Shen Xian Congee(SXC)and related targets of immune system signaling pathway based on molecular docking technology and to predict the potential mechanism of SXC in treating chronic fatigue syndrome(CFS).Established the rat model of CFS with spleenkidney yang deficiency to verify the result of molecular docking by detecting m RNA expression in vivo,and to explained the immunomodulatory mechanism of SXC on spleen-kidney yang deficiency in CFS rats.MethodsThis study is divided into two parts: molecular docking experiment and animal experiment: 1.Molecular docking experimentObtain the protein structure(control receptor)and corresponding ligand structure(control ligand)of key target proteins of NF-κB signaling pathway and Th signaling pathway from the PDB database and NCBI database.Obtain the chemical constituents of SXC(experimental receptor)from previous researches,TCMID database and TCM@Taiwan database.The Discovery Studio 3.5 software was used to study the molecular docking of the control receptor,control ligand and experimental ligand for docking operation.The combined score between control receptor and control ligand was considered as the standard to evaluate the effect strength between control receptor and experimental ligand.The signaling pathway in which the stronger control ligands is located was selected for subsequent animal experiments.2.Animal experimentAfter a week of adjustable feeding,72 SD rats were randomly divided into 6 groups(n=12): the control goup(CON),the model group(MOD),the fluoxetine hydrochloride group(positive control group,FLU),the SXC low-dose group(SXC-L),the SXC medium-dose group(SXC-M)and the SXC high-dose group(SXC-H).The experiment includes three stages for 8 weeks: modeling,intervention and evaluation.(1)Modeling: Except the CON,the other group were acctpted intervention in 4 weeks modeling.The CFS modle was prepared by exhaustion single date swinming test(EST)and double date chronic restraint stress(CRS),and the spleen and kidney yang deficiency model was established by Folium Sennae.The EST criterion is that the tip of the nose sinks into the water and can not surface for 10 s.CRS lasts 2～4h each time.Folium Sennae was given to gavage according to the standard of 1ml/100 g.After modeling,the CFS model was verified by EST and open field test(OFT)and the spleen and kidney yang deficiency model was verified by the changes of general conditions,body weight,average daily food intake and water intake.(2)Intervention: After successful modeling,6 groups of rats were gavaged according to the standard of 1ml/100g/d(the daily dosage per 100 g body weight is 1ml).SXC-L,SXC-M and SXCH were treated with different doses SXC respectively(0.42g/ml,0.83g/ml,1.66g/ml).FLU was treated with 0.21mg/ml fluoxetine hydrochloride.CON and MOD was gavaged with normal saline.Each group was gavaged once a day for 4 weeks.(3)Evaluation: During the intervention,the general condition of the rats was observed daily and measured by the semi-quantitative score scale weekly,the body weight was measesured weekly and the average daily food intake and water intake were measured on the 4th weekend of the intervention.At the end of the 1st,2nd,3rd and 4th week of the intervention,fatigue of rats were assessed by behavioral tests.At the end of the 4th week of intervention,the serum inflammatory cytokines and the m RNA expression in the signaling pathway were detected according to the results of the molecular docking experiment.Results 1.Molecular docking experimentAccording to previous researches and database,12 chemical components were summarized in SXC.Through the molecular docking software,the docking efficiency between the control protein and experimental receptor in the NF-κB signaling pathway was 90.63%,and the score was higher than or equal to the ratio of the control score was 66.67%.The docking efficiency between the control protein and experimental receptor in the Th signaling pathway was 75.00%,and the score was higher than or equal to the ratio of the control score was 45.00%.2.Animal experimentAt the end of intervention,1 rat in the SXC-L died due to misoperation.A total of 71 rats completed the experiment.The experimental results were as follows: 2.1 Modeling Results 2.1.1 The CFS modelCompared with CON,the result of EST and OFT were obviously changed in other groups(P<0.01).2.1.2 The spleen and kidney yang deficiency modelThe general semi-quantitative rating scale score,body weight,average daily food intake and water intake were statistical differences in the model groups and the CON(P<0.01).2.2 Intervention Results 2.2.1 Curative effect evaluation(1)General conditions: General semi-quantitative rating scale score: At the 4th weekend of intervention,the MOD was statistically different from the SXC-M and SXC-H(P<0.01).Body weight: At the 4th weekend of intervention,the MOD was statistically different from the FLU,SXC-M and SXC-H(P < 0.05).There was statistical differences between the FLU and SXC-M,SXC-H(P<0.05).Also,there was interaction between groups and times(P<0.01).Average daily food intake and water intake: After the intervention,the average daily food intake and average daily water intake of the MOD were statistically different from those of other groups(P<0.01).There was no statistical difference between the FLU and SXC-M,SXC-H(P>0.05).(2)Ethology Results: EST: At the 4th weekend of intervention,there were statistically significant differences between the MOD and each intervention group(P<0.01).The EST were obviously difference in SXC-M,SXC-H compared with FLU(P<0.01).There was interaction between groups and times(P<0.01).OFT: At the 4th weekend of the intervention,there were statistical differences in horizontal motion,vertical motion,grooming times and dejection amounts between MOD and other groups(P < 0.01).The horizontal motion,vertical motion,grooming times had an interaction between the groups and the time effect(P<0.05).There was no interaction in dejection amounts between the groups and the time effect(P>0.05).Tail suspension test(TST): At the 4th weekend of intervention,the time and range of the struggle in the MOD were statistically different from those in the other intervention groups except the SXC-L(P<0.01).There is no statistical difference between the FLU and SMC-L,SXCM,SXC-H(P>0.05).There was interaction between groups and times(P<0.01).2.2.2 Laboratory Indicators Results(1)Inflammatory cytokine: Group comparison: After intervention,there were statistical differences in serum IL-1β(F=39.208,P<0.01)and TNF-α(F=5.213,P<0.01)in each group.Pairwise comparison: horizontal motion: IL-1β: There was a statistical difference between the MOD and other groups(P < 0.01).There were statistical differences between the FLU and the SXC-L,SXC-M,SXC-H(P<0.01).TNF-α: There was a statistical difference between the MOD and other groups(P<0.01).There was no statistical difference between the FLU and the SXC-M,SXC-H(P>0.05).(2)m RNA expression: Group comparison: After intervention,there were statistical differences in m RNA expression of TAK1(F=11.491,P < 0.01),TAB(F=7.610,P<0.01),IKKα(F=11.119,P<0.01),IκB(F=6.162,P<0.01)and NF-κBp65(F=5.381,P<0.01),COX-2(F=1.213,P>0.05)in all group has no statistical differences.Pairwise comparison: horizontal motion: TAK1: There were a statistical difference between the MOD and the FLU,SXC-M,SXC-H(P<0.05),and the difference between FLU and the SXC-L,SXC-M,SXC-H was statistically significant(P<0.05).TAB: There were statistical differences between the MOD and the FLU,SXC-M,SXC-H(P<0.01),and the differences between the FLU and the SXC-L,SXC-M,SXC-H were statistically significant(P<0.05).IKKα: There were statistical differences between the MOD and the FLU,SXC-M,SXC-H(P<0.05),and the differences between the FLU and the SXC-L,SXC-M were statistically significant(P<0.05).IκBα: There were statistical differences between the MOD and the FLU,SXC-M,SXC-H(P<0.05),and the differences between the FLU and the SXC-L,SXC-M,SXC-H were statistically significant(P<0.05).NF-κBp65: There were statistical differences between the MOD and the FLU,SXC-H(P<0.05),and the differences between the FLU and the SXC-L were statistically significant(P<0.05).COX-2: There was no statistically significant difference between the MOD and other groups(P>0.05).Conclusion1.The results of molecular docking experiment show that: the 12 chemical components in SXC had the better binding consistency score with the control proteins in NF-κB signaling pathway.It is spectulated that the immunomodulatory effect of SXC in treatment of CFS may be related to NF-κB signaling pathway.2.The results of animal experiment show that:(1)CFS with spleen-kidney yang deficiency rat model could prepared by alternate swinming test and chronic restraint stress once a day,and gavage Folium Sennae once a day,4 weeks in a row.(2)The SXC has a positive effect in the general condition,body weight,spleen-kidney yang deficiency syndrome and fatigue symptoms of CFS rats with spleen-kidney yang deficiency syndrome.(3)The SXC can significantly reduce the IL-1β,TNF-α in CFS rats with spleen-kidney Yang deficiency syndrome and inhibit the m RNA expressions of TAK1,TAB,IKKα,IκB,NF-κBp65 in NF-κB signaling pathway.It is indicated that the SXC has effect of immunomodulatory on CFS rats through inhibiting proteins that are related NF-κB signaling pathway.But its role in COX-2 which is downstream effector proteins in NF-κB signaling pathway needs further investigation. |
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