| Objective:1.To systematically review and analyze the intervention time points and drug characteris-tics of promoting blood circulation for removing blood stasis therapy in randomized controlled trials(RCTs)applied in the acute phase of intracerebral hemorrhage(ICH).2.To explore the effects of Naoxueshu oral liquid application on neurological function and hematoma volume in rats at different time points based on literature study.3.To predict the mechanism of Naoxueshu oral liquid affecting endogenous hematoma clearance with the help of network pharmacology methods.Methods:1.RCTs on application of promoting blood circulation for removing blood stasis therapy in China National Knowledge Infrastructure Database(CNKI),Wan Fang Data,and PubMed databases,ClinicalTrials.gov,Chinese Clinical Trial Registry,and thd search time frame was from 2001 to 2020.The text types were limited to Chinese and English,with "intracerebral hemorrhage","acute phase of intracerebral hemorrhage","acute phase of hemorrhagic stroke","promoting blood circulation for removing blood stasis","blood breaking for removing blood stasis" as search terms.The intervention time points,drugs,and prescription composition in-volved in the included studies were entered into Excel to create a literature database,and SPSS was used for statistics and cluster analysis.2.SPF-grade SD rats were randomly divided into five groups:sham-operated group(group A),model group(group B),pre-administration group(group C),6 h post-modeling administra-tion group(group E),and 24 h post-modeling administration group(group G),with eight rats in each group.A rat ICH model was constructed using caudate nucleus injection of type ?collagenase.Before modeling,group C was given 1.8 mL/kg·d-1 Naoxueshu oral liquid by ga-vage for 7 days,and the remaining 4 groups were given 1.8 mL/kg·d-1 normal saline by gavage for 7 days.After modeling,groups A and B were given 1.8 mL/kg·d-1 normal saline by gavage.groups C and E were given 1.8 mL/kg·d-1 Naoxueshu oral liquid by gavage 6 h after modeling.and group G was given 1.8 mL/kg·d-1 Naoxueshu oral liquid by gavage 24 h after modeling.and rats in each group were given the drug every 24 h for a total of 3 days.Behavior of the rats was recorded and assessed by the modified neurological severity score(mNSS)at 1 d and 3 d after modeling,and brain images of the rats were obtained by magnetic resonance imaging(MRI)scanning technique.Using Sante DICOM Viewer software,the measurements were re-peated 3 times and averaged to obtain the hematoma volume and calculate the hematoma re-sorption rate by the formula.Rats were decapitated after 3 d of drug administration,and the expression of CD36 protein was observed by Western Blot and immunohistochemistry staining.3.The active pharmaceutical ingredients of herbs in Naoxueshu oral liquid were obtained with the help of the Traditional Chinese Medicine Systems Pharmacology Database and Anal-ysis Platform(TCMSP)and Pubchem database.The active pharmaceutical ingredients screen-ing and target prediction by SwissADME database and SwissTargetPrediction database.The GeneCards database and the Online Mendelian Inheritance in Man(OMIM)database were used to obtain hematoma clearance targets for ICH.The Gene Ontology(GO)functional annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed with the aid of Cytoscape software and plug-ins to initially explore the possible mechanisms of hematoma clearance in ICH by Naoxueshu oral liquidResults:1.A total of 102 RCTs were included.29 studies did not specify the intervention time,one study limited the intervention time to within 6 h of ICH onset,and 2 studies specified the time point of intervention to within 6-24 h of ICH onset.31 studies specified the intervention time point after 24 h of ICH onset.A total of 55 studies used oral medications of promoting blood circu-lation for removing blood stasis,such as herbal tonics,oral liquids or capsules.There were 40 prescriptions,68 herbs,and a total of 320 frequency of medication.Combined treatment ideas,the herbs can be clustered into four major categories,the first including rhubarb,leech,sze-chwan lovage rhizome,sanchi,red peony root,peach seed,safflower,mongolian milkvetch root,grass leaved sweetflag,cattail pollen,root of red rooted salvia,earth-worm,mirabilite,twotooth achyranthes root,the second including oriental waterplantain tuber,agaric,ground beetle,the third including tuckahoe,leonurus heterophyllus sweet,liquorice,and the fourth including baikal skullcap root,cape jasmine fruit,officinal magnolia bark,chinese angelica.2.The mNSS scores of rats on day 1 after modeling were not statistically significant in groups B,C,E and G.Compared with 1 d after modeling,the mNSS scores of rats in all groups decreased after 3 d of modeling,among which the scores of group B,C,E and G decreased significantly(P<0.05).The mNSS scores of the rats were measured on day 3 after modeling,and the scores of groups C and E were reduced compared with those of group B(P<0.05).The hematoma volumes of rats in groups B,C,E and G were compared between groups by MRI scans 1 d after modeling,and the differences were not statistically significant(P>0.05).The hematoma volumes of rats in groups C and E were smaller than group B(P<0.05)in each group at 3 d after modeling.Compared with group B,the hematoma absorption rate was signif-icantly higher in groups C and E(P<0.05).Western Blot results showed that CD36 expression were increased in groups B,C,E and G compared to group A(P<0.05),and CD36 expression were significantly higher in groups C and E compared with group B(P<0.05).Immunohisto-chemical results showed that,compared with group B,groups C,E and G showed a deeper and more positive expression of CD36 immunostaining around the hematoma under light micros-copy.Semi-quantitative analysis of AOD values showed that rats in groups C,E and G had significantly higher levels of CD36 expression around the hematoma compared to group B(P<0.01).3.After screening,Naoxueshu oral liquid contained a total of 235 candidate active phar-maceutical ingredients,of which 73 were in szechwanl ovage rhizome,28 were in rhubarb,33 were in mongolian milkvetch root,21 were in root-bark of peony,32 were in twotooth achy-ranthes root,51 were in grass leaved sweetflag,and 33 were in leech.After prediction,229 potential targets were obtained,including 40 for szechwanl ovage rhizome,66 for rhubarb,123 for mongolian milkvetch root,107 for root-bark of peony,114 for twotooth achyranthes root,116 for grass leaved sweetflag,and 45 for leech.Active ingredient-predicted target interaction networks were constructed with Cytoscape,with an average of 9.9 predicted targets per active ingredient and 2.7 active ingredients per predicted target.After screening,437 targets related to hematoma clearance in ICH were obtained,and 126 key targets were obtained by constructing an protein-protein interaction network affecting hematoma clearance in ICH with Naoxueshu oral liquid.A total of 43 biological processes,68 cellular components and 39 molecular func-tions were obtained from the GO functional annotation of 126 key targets(all P<0.05).KEGG enrichment analysis was performed on 126 key targets,and 72 signaling pathways were ob-tained(all P<0.01),and 34 major classes of signaling pathways were obtained after clustering.Combined with the results of CD3 6 study in the previous study,it was predicted that regulation of CD36 protein expression through modulation of TLR/NF-?B signaling pathway might be one of the mechanisms of Naoxueshu oral liquid to affect hematoma clearance in ICH.Conclusion:1.Naoxueshu oral liquid did not aggravate the neurological impairment of rats during the 6 h acute phase of ICH and did not lead to the enlargement of hematoma volume in rats.Nao-xueshu oral liquid can improve neurological function and promote hematoma clearance in rats when administered within 6 h during the acute phase of ICH,which is better than administering it 24 h after ICH.Naoxueshu oral liquid administered within 6 h after ICH is most beneficial for neurological recovery and hematoma clearance in rats.Naoxueshu oral liquid administered within 6 h after ICH initiated earlier hematoma clearance by increasing CD36 protein expres-sion.2.Naoxueshu oral liquid promotes hematoma clearance directly or indirectly by modulating multiple targets,with multiple signaling pathways involved.Combined with the results of the previous study,Naoxueshu oral liquid was predicted to promote endogenous hematoma clear-ance by regulating CD36 protein expression through modulation of the TLR/NF-?B signaling pathway. |
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