Clinical Efficacy And Anti-inflammatory Mechanism Of Zhongfeng Xingnao Granule In Intracerebral Hemorrhage

Intracerebral hemorrhage(ICH)is a serious cerebrovascular event that cause a high rate of disability and mortality,presenting an increasing worldwide medical and socioeconomic burden.Following ICH,multiple cascade reactions work together to induce programmed death of nerve cells and neurological deterioration.Currently,no specific therapeutics are available for the secondary damage induced by ICH in the clinic.Traditional Chinese Medicine(TCM)has its unique advantages to treat ICH.As TCM is increasingly gaining attention,their molecular mechanisms in the treatment of ICH are extensively investigated.Professor Shaohong Chen developed a TCM compound based on TCM theory,known as Zhongfeng Xingnao compound,to treat the secondary injury in ICH.The TCM Formula comprises four kinds of Chinese herbs in a certain ratio and has allowed a patent in China(Patent Number: ZL01823262.0,http://www2.soopat.com/Home/IIndex).In clinical studies,Zhongfeng Xingnao decoction(ZXD),a mixture of these four herbs prepared by the decoction and steam distillation in a certain ratio,can improve neurological outcomes of stroke patients.In addtion,ZXD can attenuate secondary brain injury in the stroke model.We conducted a multicenter,double-blind,RCT study to evaluate the therapeutic effect and safety of Zhongfeng Xingnao granule(ZXG),a granule dosage form prepared by the TCM compound,in ICH.Besides,we predicted the potential therapeutic target and signaling protein of ZXG formula via employing network pharmacological analysis.Taking this as an entry point,we will explore their molecular mechanisms in the treatment of ICH.Objectives 1.We will evaluate the clinical efficacy and safety of ZXG in ICH via a multicenter,randomized,double-blind,placebo-controlled pilot trial.2.We predicted the potential therapeutic targets and/or signaling proteins of ZXG formula via employing network pharmacological analysis.3.Taking this as an entry point,we will explore molecular mechanisms of ZXG in a collagenase-induced ICH model.Methods 1.In this clinical trial,we chose 70 patients with acute ICH to entered the clinical study.These recruited patients originate from the emergency department in the Affiliated Hospital of Chengdu University of TCM,Affiliated Hospital of Southwest Medical University,Affiliated Hospital of North Sichuan Medical College,and Sichuan Integrative Medicine Hospital.Patients were randomly allocated 1:1 to integrative therapeutic group,integrative treatment of Western medicine and ZXG(oral or nasal administration,8 mg,three times daily),and control group,Western medicine togethered with placebo.The aim is to observe the marked differences in the scores of NIHSS,ADL,and TCM syndromes,the changes in hematoma volume,and the safety between the two groups on days 7,14,21 post-treatment.2.We screen for target genes that ZXG compounds may exert therapeutic efficacy in TCMSP and Uniprot network databases.In addition,we gain human target genes related to ICH by searching several words,Cerebral hemorrhage,Intracerebral hemorrhage,and Brain hemorrhage,in the OMIM,Drugbank,Gene Cards,and Dis Ge NET gene databases and get a set of intersection genes between the two groups of genes by the Venn web tool.Successively,we enter these intersection genes in String network databases and create PPI network graphs.Finally,we screen for critical genes that may serve as the potential targets for ICH therapy by topological data analysis in Cytoscape software.Based on the P-value less than 0.05,we identify significantly enriched signaling pathways that may act on ICH by KEGG enrichment analysis in the DAVID network databases.After a comprehensive analysis of the research results of the Zhongfeng Xingnao compound in stroke,we select a well-relevant signaling pathway to verify the predictive result through animal experiments.3.Rats are randomly assigned to the ICH model group built by injecting ? collagenase into the right striatum,and the sham-operated group injected the saline.ICH model rats are randomized to the ZXG group and the control group again.In the ZXG group,the human-equivalent ZXG dose of rats was 2.48 mg/kg/day administered by oral gavage at 1.24 mg/kg twice daily for seven days.Two other groups of rats were synchronously gavaged by the same dose of saline.Within each group,we adopt NSS scores to evaluate the neurological function and Image-Pro Plus 6.0 software to measure hematoma volume on days 1,3,and 7 of treatment.To assess the therapeutic actions of ZXG,we compared the NSS score and the change in hematoma volume between groups.Furthermore,we detect perihematomal TLR4 expression by immunocytochemistry,and My D88,NF-?B,I?B?,CD36 expression via Western Blot,and TNF-?,IL-1?,IL-6,IL-10 protein levels in brain tissue and serum levels of IL-8 via Enzyme linked immunosorbent assay.Results 1.Clinical research:(1)Compared with the controls,ZXG-treated patients showed a marked reduction in NIHSS scores on days 7,14,21 post-treatment.Significant differences exist between groups(F=7.829,P=0.007).(2)Compared with western medicine alone,combination therapy with Western medicine and ZXG can improve ADL scores in ICH patients.The difference was significant between groups(F=5.428,P=0.023).(3)Compared with western medicine alone,integrated treatment of ZXG and western medicine contribute to lowing the scores of TCM syndromes(F=4.44,P=0.039)and improving the efficacy of TCM syndromes(Z=2.071,P=0.038)on day 21.(4)ZXG-treated patients exhibit a faster hematoma clearance than patients in the control group on days 14,21(F=4.485,P=0.038).(5)Throughout the study,ZXG-treated patients may not increase the risk of adverse drug reactions(ADR)versus the control group(P>0.05).2.Network pharmacology: We get 536 human target genes that ZXG compounds may act on in TCMSP and Uniprot network databases and 1645 human target genes related to ICH in gene databases.Successively,we gain 91 intersection genes between the two groups of genes by the Venn web tool and select 25 critical genes that may serve as the potential targets for ICH therapy by Cytoscape software,including TLR4,RELA,TNF-?,IL6,IL1 B,CXCL8,IL10,JUN,MAPK1,MMMP9,ICAM1.Finally, we identify 20 signaling pathways that may act on ICH in the DAVID network databases,including TLR pathway,TNF pathway,NF-?B pathway,MAPK pathway,VEGF pathway,HIF-1 pathway.Based on the anti-inflammatory effects of the Zhongfeng Xingnao compound in stroke,we select TLR4/NF-?B signaling pathway as a therapeutic target to determine ZXG anti-inflammatory mechanism in ICH.3.Experimental study:(1)ZXG-treated rats exhibit a marked reduction in NSS scores and a faster hematoma clearance versus the control group(P < 0.05).(2)Compared with the control group,the perihematomal content levels of TNF-?,IL-1?,IL-6,IL-10 showed a marked reduction in the ZXG group(P < 0.05).(3)perihematomal TLR4 expression significantly downregulated in the ZXG group versus the control group(P < 0.05).(4)Compared with the control group,perihematomal NF-?B expression showed a dramatic decrease and I?B? expression exhibited a significant increase in the ZXG group.(5)Compared with the control group,the CD36 expression in perihematomal brain tissues was significantly increased in the ZXG group on day three after administration(P<0.05).Conclusion 1.Combined treatment with ZXG and western medicine significantly reduce the NIHSS scores in ICH patients versus the control group,showing that the effects of improving neurological outcomes in the combined therapy group were superior to the control group.2.Compared with western medicine alone,integrated therapy of ZXG and western medicine dramatically increase ADL scores in ICH patients,showing that the effects of improving activities of daily living in the combined therapy group were superior to the control group.3.Compared with western medicine alone,integrated therapy of ZXG and western medicine can significantly decrease the scores of TCM syndromes in ICH patients,implying the effects of alleviating clinical symptoms in the combined therapy group were superior to the control group.4.Compared with western medicine alone,integrated therapy of ZXG and western medicine significantly facilitate hematoma clearance,suggesting the effects of promoting hematoma absorption in the combined therapy group were superior to the control group.5.Throughout the study,ZXG-treated patients have no increased risk of ADR when compared with the control group,suggesting ZXG is a safe TCM compound preparation to treat ICH.6.In this study,we find that ZXG may inhibit neuroinflammation through modulating TLR4/NF-?B signaling pathways and promote hematoma absorption via an increased expression of CD36,a scavenger receptor.