Clinical Efficacy And Experimental Study Of Shujin Jiannao Decoction In Post-operative Rehabilitation Of Spastic Cerebral Palsy

BackgroundCerebral palsy(CP)is a syndrome characterized by central motor dysfunction and abnormal posture,which is caused by the non-progressive and permanent brain damage at the time of developing fetus or infant.CP affects severely the physical and mental health of the patients.Due to the high cost of treatment,CP brings heavy financial burdens to patients'family and society.So far,surgery and rehabilitation training,medication are the main methods for the treatment of CP.Traditional Chinese medicine(TCM)which has its own unique theoretical system and treatment methods,is an important part of rehabilitative treatment of CP.Shujin Jiannao Decoction has achieved excellent clinical effects on CP while its specific role and mechanism are still unclear.Objective1.Clinical trial research:To evaluate the efficacy and safety of Shujin Jiannao Decoction in the treatment of spastic cerebral palsy(SCP).2.Network pharmacology research:To explore the molecular biological mechanism of Shujin Jiannao Decoction in treating CP,we screened the active components and targets of Shujin Jiannao Decoction and CP-related gene targets.After that,GO biological function and KEGG pathway enrichment were analyzed.In the end,the molecular docking was carried out to analysis core targets related to CP and main constituents.3.Animal experimental study:To explore the effectiveness and impact on JAK2/STAT3 signaling pathway of Shujin Jiannao Decoction,this compound Chinese drug was used to treat CP animal models based on the results of network pharmacology and previous research.Methods1.Clinical trial research:50 patients with SCP who had underwent selective posterior rhizotomy(SPR)in our hospital were selected as the study group.The control group was treated with routine rehabilitation treatment.On the basis of the routine rehabilitation treatment in the control group,the experimental group received the Chinese herb formula Shujin Jiannao Decoction.The course of treatment for the two groups is 2 months.After the treatment,the scores of GMFM-88 in zone D and zone E,TCM syndrome score,the muscle tone and muscle strength of lower limbs,Clinic spasticity index(CSI),Gross motor function classification system(GMFCS),Activities of daily living(ADL)in children with CP,Berg balance scale(BBS),Blood routine examination,liver and kidney function were applied to evaluate the improvement of motor dysfunction and safety in patients.2.Network pharmacology research:TCMSP database was used to screen the active components and targets of Shuj in Jiannao Decoction.The active components and targets were supplemented by retrieving relevant literature and Swiss Target Prediction database.In addition,Genecards database,OMIM database,KEGG database and Drugbank database were used to obtain for disease-related gene targets.Taking the intersection of targets of Shujin Jiannao Decoction and CP-related gene targets and the common targets were enriched by GO and KEGG using R language,the protein-protein interaction(PPI)network was constructed and core targets were selected through Cytoscape and STRING11.0 online platform.Finally,Auto Dock Tools and Pymol software were used to carry out molecular docking verification of the active components and core targets.3.Animal experimental study:Using classical Rice-Vannucci method to establish hypoxic-ischemic brain damage neonatal rat model,which were divided into 6 groups:blank control group(Sham),model control group(Model),minocycline control group(Positive),Low,Middle and High Shujin Jiannao Decoction dose group.The rats in each group were given corresponding intervention for one week.Hanging test,involuntary movement and Berdson score were applied to evaluate the neurological function of rats before and after treatment.After treatment,the histopathological changes of rat brain were observed by hematoxylin-eosin staining.The Content of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and interleukin-10(IL-10)in rat brain were detected by ELISA.The expression levels of JAK2,p-JAK2,p-STAT3 in the brain tissue were detected by immunohistochemistry and western blot.Results1.Clinical trial research(1)After two months of treatment,significant differences were founded between experimental group and control group in the scores of GMFM-88 in zone D and zone E,muscle tone of lower limbs,CSI,ADL,BBS by intragroup comparison(P<0.05).(2)There was no remarkable difference on the scores muscle tone of iliopsoas,quadriceps and triceps Surae,muscle strength of quadriceps and triceps Surae,CSI between two groups after treatment(P>0.05).The comparison of the scores of GMFM-88 in zone D and zone E,TCM syndrome score,muscle strength of iliopsoas,ADL,BBS of the two groups showed a significant advantage of the experimental group over the control group(P<0.05).Besides,the control group had 3 patients(13.64%)and the experimental group had 5 patients(22.73%)who undewent GMFCS decreased.(3)There were no adverse events in two groups during the treatment.2.Network pharmacology research(1)120 active components and 284 targets of Shujin Jiannao Decoction were screened by retrieving TCMSP database and the related literatures.The number of corresponding targets of CP was 1893,of which 126 are common targets with Shujin Jiannao Decoction.(2)PPI network analysis showed that AKT1,ALB,TNF,IL-6,VEGFA,JUN,EGF,CASP3,EGFR,MAPK8,TP53 and CXCL8 may be the potentially core targets.(3)The common targets were mainly involved in molecular function of response to lipopolysaccharide,response to molecule of bacterial origin,response to antibiotic,response to oxidative stress,neuron death,regulation of apoptotic signaling pathway,response to hypoxia and pathyways of the PI3K/AKT signaling pathway,AGE/RAGE signaling pathway,MAPK signaling pathway,IL-17 signaling pathway,TNF signaling pathway,HIF-1 signaling pathway,Toll-like receptor signaling pathway,p53 signaling pathway,NF-?B signaling pathway,JAK/STAT signaling pathway through GO function enrichment analysis and KEGG pathway analysis.The validity of JAK2/STAT3 signaling pathway,which was closely related to neuroinflammation,would be proved by animal experiment.(4)Molecular docking showed that the active ingredients such as quercetin,kaempferol,beta-sitosterol,stigmasterol,formononetin,baicalein had better affinity on AKT1,VEGFA,ALB and JUN.3.Animal experimental study(1)Neurological behavior:At 24h after the model establishments,the hanging test score of group Sham were significantly higher than other groups(P<0.05),while the involuntary movement and Berdson score of Sham were significantly lower than other groups(P<0.05).After one week of the model establishments,the hanging test score of group Positive,Low,Middle and High were high than group Model,the difference was significant(P<0.05).The involuntary movement of group Positive,Low,Middle were less than group Model and the difference was not significant(P>0.05).The involuntary movement of group High were high than group Model and the difference was significant(P<0.05).The Berdson score of group Positive,Low,Middle and High were less than group Model and the difference was not significant(P>0.05).Compared with group Positive,the hanging test,involuntary movement and Berdson score of group Low,Middle and High were not significantly different(P>0.05).(2)HE staining:The morphology of neurons was normal and the cell structure was complete and arranged orderly in group Sham.The structure of neurons was disordered,the intercellular space expanded and the cortical vacuolization increased greatly in group Model.Meanwhile,there were a large number of necrotic neurons and infiltrating microglia in group Model.Compared with group Model,the hypoxic-ischemic brain damage were significantly relieved in group Positive,Low,Middle and High.Pathology section in group Positive,Low,Middle and High showed necrosis of part of neurons,the intercellular space expanded,cytoplasm vacuoles and ill-defined contours of nuclear.(3)ELISA:Compared with group Model,the levels of TNF-?,IL-1? were significantly increased in group Middle and High(P<0.05).The levels of IL-10 in group Low,Middle and High were higher than than in group Model and the difference was not significant(P>0.05).Compared with group Positive,the level of IL-1? in group High significantly decreased(P<0.05)and the level of TNF-? in group Low increased markedly(P<0.05).The levels of IL-10 in group Low,Middle and High were higher than than in group Positive and the difference was not significant(P>0.05).The levels of TNF-?,IL-1? were decrease and the the levels of IL-10 were increase with incremental drug concentration of Shujin Jiannao Decoction.(4)Immunohistochemistry:The expression of JAK2 in group Sham was negative and a small amount of JAK2 protein expression can be seen in other groups.There were a small amount of p-JAK2 and p-STAT3 protein expression in group Sham.The expression of p-JAK2 and p-STAT3 in group Model,Positive,Low,Middle and High were significantly more than group Sham.(5)Western blot:Compared with group Sham,after one week of the treatment,the expression of JAK2,p-JAK2 and p-STAT3 were significantly increased in group Model,Positive,Low,Middle and High(P<0.05).The expression of JAK2,p-JAK2 and p-STAT3 in the brain tissue were significantly reduced in group Sham,Positive,Low,Middle and High compared with group Model(P<0.05).For the expression of JAK2,p-JAK2 and p-STAT3,there were no significant difference between Group Positive and Group Low,Middle,High(P>0.05).There were no remarkable difference on the expression of JAK2,p-JAK2 and p-STAT3 between Group Low,Middle,High(P>0.05).Conclusions1.Shujin Jiannao Decoction is effective,safe for the treatment of SCP patients who had underwent SPR.2.The network pharmacology research studies have shown that the main mechanism of Shujin Jiannao Decoction in treating CP is involved to many potential active components,signaling pathways and multiple core targets.JAK2/STAT3 signaling pathway,which involves in the regulation of neuroinflammation,plays an important role in the treatment of CP with Shujin Jiannao Decoction.3.Shujin Jiannao Decoction can alleviate neuroinflammation and brain injury,improve neurological deficit in model rats,the mechanism may be related to the down-regulation of the JAK2/STAT3 signaling pathway.