OBJECTIVES: To explore the effects of cannabidiol on endoplasmic reticulum stress and neuronal apoptosis in rats with multiple concussions (MCC). METHODS: SD rats were randomized into sham group, MCC group, 1% tween20 (TW) treatment group, and low-dose (10 mg/kg) and high-dose (40 mg/kg) cannabidiol treatment groups. In all but the sham group, MCC models were established using a metal pendulum percussion device, after which the rats received daily intraperitoneal injections of the corresponding agents for 2 weeks. The expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-Akt and pro-caspase-3 in the brain tissue of the rats were detected with qRT-PCR, Western blotting and immunofluorescence staining. The core targets of cannabidiol in treatment of traumatic brain injury (TBI) were identified by network pharmacology analysis, and molecular docking was carried out to simulate the interaction of cannabidiol with the factors related to endoplasmic reticulum stress and apoptosis. RESULTS: Compared with the sham-operated rats, the rat models of MCC showed significantly increased mRNA expressions of PERK, eIF2α and CHOP and protein expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-AKT and pro-caspase-3 in the cerebral cortex. CBD treatment, especially at the high dose, obviously increased the expression of p-Akt and lowered the expression levels of the other factors tested in the rat models. Network pharmacology analysis indicated interactions of the core targets of CBD with the factors related to endoplasmic reticulum stress and TBI, and molecular docking study showed a high binding energy of CBD with multiple factors pertaining to endoplasmic reticulum stress and apoptosis. CONCLUSIONS MCC induce endoplasmic reticulum stress and apoptosis in rat brain tissues, for which CBD, especially at a high dose, provides neuroprotective effects by inhibiting endoplasmic reticulum stress and cell apoptosis.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Apoptosis/drug effects* ; Rats, Sprague-Dawley ; Activating Transcription Factor 4/metabolism* ; Transcription Factor CHOP/metabolism* ; Rats ; Eukaryotic Initiation Factor-2/metabolism* ; Signal Transduction/drug effects* ; eIF-2 Kinase/metabolism* ; Cannabidiol/pharmacology* ; Neurons/metabolism* ; Brain Concussion/metabolism* ; Male ; Molecular Docking Simulation

AIM:To determine if scutellarin(Scu)provides neuroprotection by reducing neuronal apoptosis in rats subjected to middle cerebral artery occlusion(MCAO)via the inhibition of the JAK2/STAT3 signalling pathway.METHODS:Proteins linked to Scu and ischaemic stroke-induced neuronal apoptosis were identified using the Swiss Tar-get Prediction,PharmMapper,OMIM,and GeneCards databases.Intersecting targets were identified through Venn analy-sis.Protein-protein interaction networks were visualised utilising Cytoscape software,and principal targets were identi-fied.Enrichment analyses of GO functions and KEGG pathways were conducted utilising the Metascape database.Molecu-lar docking of Scu with core targets was performed utilising AutoDock Vina.The neuroprotective effects of Scu were as-sessed in MCAO rats using Zea Longa scoring and the suspension test.JAK2/STAT3 phosphorylation levels and apoptosis-related proteins[cleaved caspase-3(C-caspase-3),caspase-3,Bax,and Bcl-2]were assessed using Western blot and im-munofluorescence staining.The JAK2-specific inhibitor AG490 was employed to further investigate the role of the JAK2/STAT3 signaling pathway.RESULTS:Network pharmacology analysis revealed 832 shared targets,with pathways en-riched in tumor-associated pathways,the JAK/STAT signalling pathway,and the HIF-1 signalling pathway.Molecular docking revealed robust binding affinities of Scu with the ten principal targets.Behavioural assessments utilising Zea Lon-ga scoring and the suspension test demonstrated that Scu markedly enhanced neurological recovery in MCAO rats.Western blot and immunofluorescence analyses demonstrated that phosphorylation levels of JAK2 and STAT3,along with the ex-pression of C-caspase-3,Bax,and Bcl-2,were markedly elevated in the MCAO group relative to the sham group(P<0.05).Post Scu treatment,phosphorylation levels of JAK2 and STAT3,along with C-caspase-3 and Bax expression,were markedly diminished,whereas Bcl-2 expression and fluorescence intensity were substantially increased(P<0.05).In the combined AG490 and Scu treatment group(MCAO+Scu+AG490),the phosphorylation levels of JAK2 and STAT3,as well as the expression of C-caspase-3 and Bax,exhibited no significant difference when compared to the Scu-alone group(P>0.05).Bcl-2 expression and fluorescence intensity were markedly reduced in the combined AG490 and Scu treatment group relative to the Scu-alone group(P<0.05).CONCLUSION:Scu seems to provide neuroprotection in ischaemic stroke by reducing neuronal apoptosis through the inhibition of the JAK2/STAT3 signalling pathway.

AIM:To determine if scutellarin(Scu)provides neuroprotection by reducing neuronal apoptosis in rats subjected to middle cerebral artery occlusion(MCAO)via the inhibition of the JAK2/STAT3 signalling pathway.METHODS:Proteins linked to Scu and ischaemic stroke-induced neuronal apoptosis were identified using the Swiss Tar-get Prediction,PharmMapper,OMIM,and GeneCards databases.Intersecting targets were identified through Venn analy-sis.Protein-protein interaction networks were visualised utilising Cytoscape software,and principal targets were identi-fied.Enrichment analyses of GO functions and KEGG pathways were conducted utilising the Metascape database.Molecu-lar docking of Scu with core targets was performed utilising AutoDock Vina.The neuroprotective effects of Scu were as-sessed in MCAO rats using Zea Longa scoring and the suspension test.JAK2/STAT3 phosphorylation levels and apoptosis-related proteins[cleaved caspase-3(C-caspase-3),caspase-3,Bax,and Bcl-2]were assessed using Western blot and im-munofluorescence staining.The JAK2-specific inhibitor AG490 was employed to further investigate the role of the JAK2/STAT3 signaling pathway.RESULTS:Network pharmacology analysis revealed 832 shared targets,with pathways en-riched in tumor-associated pathways,the JAK/STAT signalling pathway,and the HIF-1 signalling pathway.Molecular docking revealed robust binding affinities of Scu with the ten principal targets.Behavioural assessments utilising Zea Lon-ga scoring and the suspension test demonstrated that Scu markedly enhanced neurological recovery in MCAO rats.Western blot and immunofluorescence analyses demonstrated that phosphorylation levels of JAK2 and STAT3,along with the ex-pression of C-caspase-3,Bax,and Bcl-2,were markedly elevated in the MCAO group relative to the sham group(P<0.05).Post Scu treatment,phosphorylation levels of JAK2 and STAT3,along with C-caspase-3 and Bax expression,were markedly diminished,whereas Bcl-2 expression and fluorescence intensity were substantially increased(P<0.05).In the combined AG490 and Scu treatment group(MCAO+Scu+AG490),the phosphorylation levels of JAK2 and STAT3,as well as the expression of C-caspase-3 and Bax,exhibited no significant difference when compared to the Scu-alone group(P>0.05).Bcl-2 expression and fluorescence intensity were markedly reduced in the combined AG490 and Scu treatment group relative to the Scu-alone group(P<0.05).CONCLUSION:Scu seems to provide neuroprotection in ischaemic stroke by reducing neuronal apoptosis through the inhibition of the JAK2/STAT3 signalling pathway.

Objective:To explore the impact of scutellarin on the levels of phosphatidylinositol 3-kinase(P13K)and phosphorylated P13K(p-PI3K)in activated microglia.Methods:A rat cerebral ischemia model was made by middle cerebral artery occlusion(MCAO)method.SD rats were randomly divided into three groups:sham operation group(Sham),cerebral ischemia group(MCAO),and cerebral ischemia with scutellarin group(MCAO+S).A model of ischemia-hypoxia injury of BV2 microglia was established by glucose-oxygen deprivation(OGD)and randomly assigned as control group(Control),OGD group and OGD+scutellarin group(OGD+S).Changes in PI3K and p-PI3K expres-sion in microglia were assessed using double immunofluorescence staining and Western Blot.Results:Immunofluores-cence staining and Western Blot analyses revealed a significant increase in p-PI3K levels in activated microglia both in vivo and in vitro(P＜0.05).Furthermore,treatment with scutellarin led to a further elevation in p-PI3K(P＜0.05).However,there were no significant alterations observed in PI3 K expression among the groups(P＞0.05).Conclusion:Scutellarin may play a positive role in the treatment of cerebral ischemia by up-regulating the phosphorylation level of P13K in activated microglia.

Objective:To investigate the effects of cannabidiol(CBD)on the NOD-like receptor protein 3(NLRP3)inflammasome in the brains of rats with multiple cerebral concussions(MCC).Methods:Rats were subjec-ted to the MCC model and divided into Sham,MCC,vehicle(MCC+TW),CBD-L(10 mg/kg),and CBD-H(40 mg/kg)groups.Immunofluorescence double staining was used to observe changes in NLRP3 and microglial cells in the brain,and Western Blot was performed to detect the expression changes of the NLRP3 inflammasome.Results:A sig-nificant increase in lectin-positive microglial cells of the cortex with enlarged cell bodies and elevated immunofluores-cence intensity of NLRP3 in the activated microglial cells was revealed by immunofluorescence double staining following MCC(P＜0.05).The immunofluorescence intensity of NLRP3 in the activated microglial cells was downregulated by the administration of CBD,with a more pronounced effect observed in the CBD-H group compared to the CBD-L group(P＜0.05).The expression of NLRP3,caspase-1,and apoptosis-associated speck-like protein(ASC)in the cortex,hippocampus,and basal ganglia of rats following MCC was significantly increased,as shown by Western Blot analysis(P＜0.05),and cortical areas are more elevated.The expression of these proteins in different brain regions was reduced by CBD-10 and CBD-40 intervention(P＜0.05).Conclusion:Cannabidiol can reduce the inflammatory response of multiple cerebral concussions rats through NLRP3 inflammasome and protect nerve tissue.

Objective:Investigating the effect of scutellarin on the expression of silent information regulator 1/nucle-ar factor κB(SIRT1/NF-κB)signaling pathway in microglia of cerebral ischemic rat.Methods:Male SD rats were de-vided into sham group,middle cerebral artery occlusion group(MCAO group),MCAO+scutellarin treatment group(MCAO+S group)randomly.Western Blot and immunofluorescence were used to detect the expression of SIRT1 and NF-κB(p65)and phosphorylation of NF-κB(p-p65)of ischemic cortex of rats.Results:Results of Western Blot and immunofluorescence showed that scutellarin pretreatment significantly increased the expression of SIRT1 and reduced phosphorylation of NF-κB p65(P＜0.05).Conclusion:Scutellarin could regulate SIRT1/NF-κB signaling pathway in microglia of cerebral ischemic rat.

Objective:Investigating the effect of scutellarin on the expression of silent information regulator 1/nucle-ar factor κB(SIRT1/NF-κB)signaling pathway in microglia of cerebral ischemic rat.Methods:Male SD rats were de-vided into sham group,middle cerebral artery occlusion group(MCAO group),MCAO+scutellarin treatment group(MCAO+S group)randomly.Western Blot and immunofluorescence were used to detect the expression of SIRT1 and NF-κB(p65)and phosphorylation of NF-κB(p-p65)of ischemic cortex of rats.Results:Results of Western Blot and immunofluorescence showed that scutellarin pretreatment significantly increased the expression of SIRT1 and reduced phosphorylation of NF-κB p65(P＜0.05).Conclusion:Scutellarin could regulate SIRT1/NF-κB signaling pathway in microglia of cerebral ischemic rat.

Objective:To explore the potential molecular mechanism of Erigeron breviscapus in the prevention and treatment of ischemic stroke through network pharmaco-molecular docking.Methods:The Chinese Herbal Medicine Systematic Pharmacology Platform(TCMSP)database provided active ingredients and potential targets of Erigeron breviscapus.Ischemic stroke-related targets were searched through the Online Mendelian Inheritance in Man(OMIM)database,the bioinformatics and chemoinformatics(DrugBank)database and human gene comprehensive database(GeneCards).The targets criteria were de-weighted by the Protein Data Bank(Uniprot)and imported into the Venny online platform to obtain the intersecting targets of both.The intersecting targets were visualized by STRING database and Cytoscape 3.7.1 software for protein-protein interaction(PPI),followed by the enrichment analysis of intersection targets for gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.AutoDock Vina1.5.6 software was used to verify the molecular docking of key active ingredients and core targets and realize the vi-sualization of docking results.Results:Eleven active ingredients and 176 targets were obtained.There were 690 targets ischemic stroke-related targets and 69 intersection targets.Through PPI network,10 core genes were screened according to the degree value,including tumor necrosis factor(TNF),interleukin-6(IL-6),serine/threonine protein kinase 1(AKT1),interleukin-1β(IL-1β)and vascular endothelial growth factor A(VEGFA).KEGG enrichment included the advanced glycation end products-receptor(AGE-RAGE)signaling pathway,interleukin-17(IL-17)signaling pathway,tumor necrosis factor(TNF)signaling pathway,etc.The top 3 active ingredients and the top 5 target proteins were selected according to the degree value,and the molecular docking results demonstrated a considerable binding ability.Conclusion:Erigeron breviscapus in the treatment of ischemic stroke may work through multiple active ingredients,such as quercetin,kakaferol,and luteolin,which act on TNF,IL-6,AKT1,IL-1β,and VEGFA,and through a varie-ty of signaling pathways such as IL-17,TNF,etc.showing the characteristics of multi-components,multi-targets,and multi-pathways.

【Objective】 To investigate the effect of transfusion-transmitted Zika virus (ZIKV) on the expression of non-coding circular RNA (hsa_circ_0001613) and the role of hsa_circ_0001613 in Zika virus replication. 【Methods】 Human adenocarcinomic alveolar basal epithelial cells (A549) were seeded on a 12-well plate at 1.8×10⁵/ well and infected with ZIKV at 0.05 MOI. The Total RNAs were isolated every day for 5 days after infection, and the relative expression level of hsa_circ_0001613 was detected by qRT-PCR. In addition, 10nM siRNA-hsa_circ_0001613 was transfected into 2×10⁵/ well A549 cells to specifically knock down the expression level of hsa_circ_0001613. 24h later, the cells were infected with ZIKV (MOI=0.05). Total RNAs were isolated at day 1-5 post-infection, proteins were extracted 96h post-infection. ZIKV replication, relative host antiviral gene expression, and interferon stimulated response element (ISRE) activity were tested using qRT-PCR, western blot and dual luciferase assay, respectively. 【Results】 The relative expression of hsa_circ_0001613 decreased significantly after 1-5 days of ZIKV infection. Knockdown of hsa_circ_0001613 inhibited ZIKV replication. Meanwhile, hsa_circ_0001613 knockdown significantly upregulated IFN-α/β and its downstream interferon-stimulated genes (ISGs) expression, also increased ISRE activity. 【Conclusion】 ZIKV infection significantly suppressed hsa_circ_0001613 expression in A4549 cells. Preliminary study indicated that hsa_circ_0001613 knockdown inhibited ZIKV replication possibly through activating type-Ⅰ IFN signaling pathway as showed by increased ISGs expression and ISRE activity.

Objective:To investigate the effect of Wuwei-Xiaodu Decoction in treating intra-articular calcaneal fractures after open reduction and internal fixation and its effect on hemodynamics. Methods:A total of 88 intra-articular calcaneal fractures patients admitted to Langfang People's Hospital and the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine from April of 2016 to February of 2019 were selected and randomly divided into the control group and the observation group (44 patients in each group) by the method of random number table. Both groups received open reduction and internal fixation for intra-articular calcaneal fractures. The control group received conventional treatment after surgery. The observation group were treated with Wuwei-Xiaodu Decoction on the basis of treatment in the control group. Both groups were treated for 12 weeks. The Gissane Angle, Bohler Angle and calcaneal height were detected by X-ray and CT. The hemodynamic analyzer was used to determine ESR, Fibrinogen (Fb), and whole blood viscosity. The recovery of foot function was evaluated by using the Ankle-posterior foot scoring system of the American Orthopaedic Association, and the fracture healing time and complications were recorded. Results:After treatment, the Gissane Angle (130.01°± 15.76° vs. 121.33°± 14.33°, t=2.793), Bohler Angle (33.04°± 5.03° vs. 28.93°± 4.31°, t=4.189) and calcaneal height (44.13 ± 6.13 mm vs. 40.06 ± 5.87 mm, t=6.556) in the observation group were significantly higher than those in the control group ( P<0.01). The fracture healing time (66.85 ± 8.17 d vs. 78.04 ± 9.44 d, t=5.945) in the observation group was significantly lower than that of the control group ( P<0.01). After treatment, serum ESR (13.65 ± 1.94 mm/h vs. 18.99 ± 2.30 mm/h, t=11.772), Fb (301.83 ± 32.04 g/L vs. 319.85 ± 33.62 g/L, t=2.536) levels and whole blood viscosity (7.04 ± 0.82 mPa?s vs. 8.34 ± 0.92 mPa?s, t=6.997) in the observation group were significantly lower than those in the control group ( P<0.01). The effective rate of foot function recovery 6 months after operation in the observation group was 97.7% (43/44) and 81.8% (36/44) in the control group, and the difference between the two groups was statistically significant ( χ2=4.456, P=0.035). The complication rate was 4.6% (2/44) in the observation group and 20.5% (9/44) in the control group, and the difference between the two groups was statistically significant ( χ2=5.091, P=0.024). Conclusions:The Wuwei-Xiaodu Decoction can promote the recovery of bone shape after open reduction and internal fixation of intra-articular calcaneal fractures, reduce the complications after operation, promote fracture healing, and improve the hemodynamics.