ObjectiveTo investigate whether Huanglian Jiedutang （HLJD） and its major active constituents （geniposide， baicalin， and berberine） can inhibit the inflammatory response of BV2 cells under lipopolysaccharide （LPS） stimulation via the high-mobility group protein B1 （HMGB1）/Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway， and to explore differences in therapeutic efficacy among the three monomers， their combined formula， and HLJD under equal content ratios. MethodsBV2 microglial cells were used as the primary experimental model. Cell viability was assessed using the cell counting kit-8 （CCK-8） method to examine the effects of different concentrations of dimethyl sulfoxide （DMSO， 0.8%， 0.4%， 0.2%， 0.1%， and 0.05%） on cell viability. IncuCyte was employed to monitor the growth of cells under different concentrations of HLJD （200， 100， 50， 25， 12.5， 6.25 mg·L^-1）. Nitric oxide （NO） assay was used to screen the optimal HLJD concentration. High-performance liquid chromatography （HPLC） determined the content of geniposide， baicalin， and berberine in HLJD， and experimental groups were subsequently established according to the relative proportions of these constituents. CCK-8 assay evaluated cell viability under different treatments. Enzyme-linked immunosorbent assay （ELISA） measured levels of inflammatory factors （TNF-α， IL-1β， IL-6， IL-10） in the supernatant. Flow cytometry assessed the effects of treatments on M1-type polarization of BV2 cells. Western blot determined the expression levels of HMGB1， TLR4， and NF-κB-related proteins. ResultsCompared with the blank group， DMSO at concentrations ≤0.2% did not affect cell viability within 48 h. BV2 cell growth plateaued at 24 h after treatment with 200 mg·L^-1 HLJD. Under stimulation with 2 mg·L^-1 LPS， this concentration of HLJD effectively reduced NO release， and 6 h pre-treatment had a stronger inhibitory effect on NO than direct administration. HPLC results showed that 1 mg of HLJD freeze-dried powder contained approximately 24 μg of geniposide， 15 μg of baicalin， and 30 μg of berberine. Based on these ratios， experimental groups were blank， LPS （2 mg·L^-1）， HLJD （200 mg·L^-1）， monomer combination， geniposide （4.8 mg·L^-1）， baicalin （3 mg·L^-1）， and berberine （6 mg·L^-1）. The monomer combination group consisted of all three active constituents dissolved together. LPS and HLJD or its active constituents did not affect cell viability compared with the blank group. LPS significantly increased TNF-α， IL-1β， IL-6， and IL-10 in the supernatant （P<0.01）. HLJD and its active constituents significantly reduced pro-inflammatory factors TNF-α， IL-1β， and IL-6 （P<0.05， P<0.01） while upregulating anti-inflammatory IL-10 （P<0.01）， with the monomer combination showing the strongest effect （P<0.05， P<0.01）. Compared with the blank group， LPS significantly increased the proportion of CD80⁺CD86⁺ （M1-type） BV2 cells （P<0.01）. HLJD and its constituents partially inhibited M1 polarization （P<0.05， P<0.01）， with the monomer combination exhibiting the most pronounced effect （P<0.05， P<0.01）. Compared with the blank group， LPS upregulated HMGB1， TLR4， and NF-κB-related proteins （P<0.01）， whereas HLJD and its active constituents significantly reduced their expression （P<0.05， P<0.01）， with the monomer combination having the strongest regulatory effect （P<0.05， P<0.01）. ConclusionHLJD and its major active constituents （geniposide， baicalin， berberine） can inhibit LPS-induced inflammatory responses in BV2 cells. The combination of the three active constituents demonstrates the most potent anti-inflammatory effect， significantly attenuating M1-type polarization of BV2 cells via the HMGB1/TLR4/NF-κB signaling pathway.

Objective To investigate the mechanism of Wenhe Decoction in the treatment of febrile seizures through network pharmacology based on NLRP3/Caspase-1/GSDMD signaling pathway;To conduct experimental verification.Methods The active components and targets of Wenhe Decoction were retrieved and screened through TCMSP,BATMAN-TCM,PubChem databases and SwissADME platform.The disease targets of febrile seizures were found in GenCards,OMIM and DisGeNET databases.The intersection targets of Wenhe Decoction and the disease and the active components corresponding to the intersection targets were imported into Cytoscape 3.7.2 software to construct the Chinese materia medica-active components-targets network.The intersection targets were submitted to the STRING database to construct a protein-protein interaction network,and then the intersection targets were imported into the Metascape database for GO and KEGG pathway enrichment analysis.The febrile seizures rat model was established,and Wenhe Decoction of 4.05,8.1 and 16.2 g/kg were given respectively by gavage for 21 days.The rats were placed in batches in(45±0.5)℃constant temperature water bath to induce convulsive seizures,and the convulsive latency time and convulsive duration of rats were recorded.The behavioral differences of mice were observed.The morphology of hippocampal tissue were observed by HE and Nissl staining.The ROS content of hippocampal tissue was detected by DHE fluorescent probe technology.The serum ATP,GABA,Glu,Caspase-1,GSDMD,IL-1β and IL-18 contents were detected by ELISA,and the expression of NLRP3 inflammasome-related protein in hippocampal tissue was detected by Western blot.Results Network pharmacology analysis obtained 98 active components of Wenhe Decoction and 1 838 targets.162 targets were obtained by intersecting with disease targets,the core components for the treatment of febrile seizures were β-sitosterol,quercetin,luteolin,trans-squalene,sitosterol,saponin,etc.,and the core targets were EGFR,TNF,JUN,MTOR,etc.,and mainly through the regulation of inflammatory response,apoptosis,mitochondrial function and energy metabolism,mediating anti-inflammatory pathways such as PI3K-Akt signaling pathway and calcium signaling pathway to exert anticonvulsant effects.The experimental results showed that Wenhe Decoction could prolong the convulsive latency and shorten the duration of convulsions in febrile seizures model rats,decrease the level of convulsions,and the pathological damage of hippocampal tissue was improved and damaged neurons were repaired.The serum contents of ROS,Glu,Caspase-1,GSDMD,IL-1β and IL-18 were significantly reduced,and ATP and GABA contents significantly increased.The protein expressions of NLRP3,ASC,pro-Caspase-1,pro-IL-1β and pro-IL-18 in hippocampal tissue significantly decreased.Conclusion Wenhe Decoction may intervene in febrile seizures rats through NLRP3/Caspase-1/GSDMD signaling pathway,inhibit pyroptosis to reduce the occurrence of neuroinflammation,and then affect the balance of neurotransmitters Glu and GABA to play a role in anti-febrile seizures and prevent brain tissue damage.

Objective To investigate the mechanism of Wenhe Decoction in the treatment of febrile seizures through network pharmacology based on NLRP3/Caspase-1/GSDMD signaling pathway;To conduct experimental verification.Methods The active components and targets of Wenhe Decoction were retrieved and screened through TCMSP,BATMAN-TCM,PubChem databases and SwissADME platform.The disease targets of febrile seizures were found in GenCards,OMIM and DisGeNET databases.The intersection targets of Wenhe Decoction and the disease and the active components corresponding to the intersection targets were imported into Cytoscape 3.7.2 software to construct the Chinese materia medica-active components-targets network.The intersection targets were submitted to the STRING database to construct a protein-protein interaction network,and then the intersection targets were imported into the Metascape database for GO and KEGG pathway enrichment analysis.The febrile seizures rat model was established,and Wenhe Decoction of 4.05,8.1 and 16.2 g/kg were given respectively by gavage for 21 days.The rats were placed in batches in(45±0.5)℃constant temperature water bath to induce convulsive seizures,and the convulsive latency time and convulsive duration of rats were recorded.The behavioral differences of mice were observed.The morphology of hippocampal tissue were observed by HE and Nissl staining.The ROS content of hippocampal tissue was detected by DHE fluorescent probe technology.The serum ATP,GABA,Glu,Caspase-1,GSDMD,IL-1β and IL-18 contents were detected by ELISA,and the expression of NLRP3 inflammasome-related protein in hippocampal tissue was detected by Western blot.Results Network pharmacology analysis obtained 98 active components of Wenhe Decoction and 1 838 targets.162 targets were obtained by intersecting with disease targets,the core components for the treatment of febrile seizures were β-sitosterol,quercetin,luteolin,trans-squalene,sitosterol,saponin,etc.,and the core targets were EGFR,TNF,JUN,MTOR,etc.,and mainly through the regulation of inflammatory response,apoptosis,mitochondrial function and energy metabolism,mediating anti-inflammatory pathways such as PI3K-Akt signaling pathway and calcium signaling pathway to exert anticonvulsant effects.The experimental results showed that Wenhe Decoction could prolong the convulsive latency and shorten the duration of convulsions in febrile seizures model rats,decrease the level of convulsions,and the pathological damage of hippocampal tissue was improved and damaged neurons were repaired.The serum contents of ROS,Glu,Caspase-1,GSDMD,IL-1β and IL-18 were significantly reduced,and ATP and GABA contents significantly increased.The protein expressions of NLRP3,ASC,pro-Caspase-1,pro-IL-1β and pro-IL-18 in hippocampal tissue significantly decreased.Conclusion Wenhe Decoction may intervene in febrile seizures rats through NLRP3/Caspase-1/GSDMD signaling pathway,inhibit pyroptosis to reduce the occurrence of neuroinflammation,and then affect the balance of neurotransmitters Glu and GABA to play a role in anti-febrile seizures and prevent brain tissue damage.

Objective:To explore the effects of miR-125b on the proliferation, invasion and migration of pancreatic cancer cells by targeting SMYD2 signaling pathway.Methods:The expression of miRNA-125b in Aspc-1 and BxPC-3 lines of pancreatic cancer cells were detected. miRDB, ENCORI and TargetScan databases were used to predict the potential target genes of miRNA-125b. The downstream target genes of miRNA-125b were identified by qPCR assay and double luciferase reporter gene assay. Western blot analysis was performed to detect SMYD2 protein expression after transfection with miRNA-125b inhibitor. EdU staining, Annexin V-FITC/PI assay and Annexin V-FITC/PI assay were used to detect the effects of miRNA-125b inhibitor transfection and simultaneous transfection of miRNA-125b and SMYD2 inhibitor on cell proliferation, clonogenesis and apoptosis.Results:The expression level of miRNA-125b in pancreatic cancer cell lines was higher than that in normal pancreatic duct cells ( P<0.05). The downstream target gene of miRNA-125b was identified as SMYD2 by qPCR assay and double luciferase reporter gene assay. The expression of SMYD2 protein in miR-125b inhibitor group was higher than that in NC group ( P<0.01). EdU cell proliferation assay showed that the number of miRNA-125b positive cells in inhibitor group was lower than that in NC group and Inhibitor NC group ( P<0.05). The number of clones in miR-125b inhibitor+si-SMYD2 group was more than that in miR-125b inhibitor group ( P<0.01). Annexin V-FITC/PI assay showed that the apoptosis number of cell cells in miR-125b inhibitor+si-SMYD2 group was lower than that in miR-125b inhibitor group ( P<0.01) . Conclusion:miRNA-125b is highly expressed in pancreatic cancer cells, and can directly affect the expression of SMYD2 gene, thereby promoting the proliferation and inhibiting the apoptosis of pancreatic cancer cells.

BACKGROUND:Current strategies for the treatment of the corticospinal tract after spinal cord injury mainly focus on exercise rehabilitation,drug therapy,transcranial magnetoelectric stimulation,endogenous regulation such as transcription factors and specific signaling pathways.Among them,transcription factors and their specific signaling pathways are the key factors regulating the axonal regeneration of corticospinal tract after spinal cord injury.A large number of preclinical studies have confirmed that the synergy between transcription factors and their signaling pathways has a significant regulatory effect on the axonal regeneration of neurons in the corticospinal tract after spinal cord injury.Therefore,it has broad application prospects to explore new combination therapy strategies targeting transcription factors and specific signaling pathways for spinal cord injury.OBJECTIVE:To systematically summarize the regulatory effects of transcription factors and their signaling pathways on the axonal regeneration of neurons in the corticospinal tract after spinal cord injury and the underlying molecular mechanisms,and explore the application of combined therapy strategies targeting transcription factors and signaling pathways in the neuroplasticity of the corticospinal tract after spinal cord injury,in order to provide a new combination strategy for the treatment of spinal cord injury.METHODS:The search terms included"spinal cord injury,axon regeneration,transcription factors,signaling pathway,corticospinal tract,central nervous system,synergistic system,neuroprotective system"in Chinese and English.A literature retrieval was conducted in WanFang,Web of Science,and PubMed for relevant literature published from database inception to September 2024.Finally,101 articles were included for analysis and summary.RESULTS AND CONCLUSION:(1)The article outlines the biological properties and intervention strategies for axonal regeneration of the corticospinal tract after spinal cord injury,analyses the reasons for focusing on the corticospinal tract after spinal cord injury,and elucidates the response and possibility of regeneration of the corticospinal tract after spinal cord injury.(2)In this study,the combined regulatory strategy of transcription factors centered on Krüppel-like factor 6,Krüppel-like factor 7,and neuronal restriction silencing factor can significantly promote the axonal regeneration of neurons in the corticospinal tract after spinal cord injury.(3)The phosphatidylinositol 3-kinase-protein kinase B-rapamycin target protein signaling pathway and Wnt5a pathway are the classical signaling pathways fortranscription factors to regulate the axonal regeneration of corticospinal tract neurons,and the combined treatment strategy can effectively promote the axonal regeneration and functional reconstruction of corticospinal tract neurons after spinal cord injury.(4)This article discusses the combined treatment strategies of transcription factors and specific signaling pathways in a comprehensive and detailed manner,such as Krüppel-like factor 6 combined with signal transducer and activator of transcription 3,Krüppel-like factor 7 combined with SOX11 transcription factor,combined inhibition of phosphatase and tensin homologs and neuronal restriction silencing factor,etc.,to exert a synergistic effect and promote the axonal regeneration of corticospinal tract neurons after spinal cord injury,which are significantly better than those of single treatment.It can effectively improve functional recovery and provide a reference scheme for the future treatment of axonal regeneration of corticospinal tract neurons after spinal cord injury.However,the specific mechanism of the combination therapy still needs to be further studied,and the current combination strategy is only widely used in animal models but not in clinical practice.(5)The combined therapy strategy based on transcription factors and specific signaling pathways has a significant therapeutic effect on the axonal regeneration of corticospinal tract neurons after spinal cord injury,and it is necessary to further explore the molecular mechanism of joint regulation in the future,in order to provide an effective combined therapy strategy for the rehabilitation and functional reconstruction of spinal cord injury.

Objective:To explore the effects of miR-125b on the proliferation, invasion and migration of pancreatic cancer cells by targeting SMYD2 signaling pathway.Methods:The expression of miRNA-125b in Aspc-1 and BxPC-3 lines of pancreatic cancer cells were detected. miRDB, ENCORI and TargetScan databases were used to predict the potential target genes of miRNA-125b. The downstream target genes of miRNA-125b were identified by qPCR assay and double luciferase reporter gene assay. Western blot analysis was performed to detect SMYD2 protein expression after transfection with miRNA-125b inhibitor. EdU staining, Annexin V-FITC/PI assay and Annexin V-FITC/PI assay were used to detect the effects of miRNA-125b inhibitor transfection and simultaneous transfection of miRNA-125b and SMYD2 inhibitor on cell proliferation, clonogenesis and apoptosis.Results:The expression level of miRNA-125b in pancreatic cancer cell lines was higher than that in normal pancreatic duct cells ( P<0.05). The downstream target gene of miRNA-125b was identified as SMYD2 by qPCR assay and double luciferase reporter gene assay. The expression of SMYD2 protein in miR-125b inhibitor group was higher than that in NC group ( P<0.01). EdU cell proliferation assay showed that the number of miRNA-125b positive cells in inhibitor group was lower than that in NC group and Inhibitor NC group ( P<0.05). The number of clones in miR-125b inhibitor+si-SMYD2 group was more than that in miR-125b inhibitor group ( P<0.01). Annexin V-FITC/PI assay showed that the apoptosis number of cell cells in miR-125b inhibitor+si-SMYD2 group was lower than that in miR-125b inhibitor group ( P<0.01) . Conclusion:miRNA-125b is highly expressed in pancreatic cancer cells, and can directly affect the expression of SMYD2 gene, thereby promoting the proliferation and inhibiting the apoptosis of pancreatic cancer cells.

Objective:To compare the effects of propofol and dexmedetomidine for sedation on the outcome in mechanically ventilated patients with severe pulmonary infection.Methods:Patients with severe pulmonary infection (Clinical Pulmonary Infection Score >7) requiring mechanical ventilation from the Medical Information Mart for Intensive Care Ⅳ database between 2008 and 2020 were selected and divided into propofol group and dexmedetomidine group based on the sedative agent used. The primary outcome was all-cause in-hospital mortality, and secondary outcomes included 90-day all-cause mortality and duration of mechanical ventilation. Inverse probability of treatment weighting (IPTW) was used to adjust for baseline confounders, and logistic regression and linear regression were applied to analyze the effects of the two sedative drugs on the outcomes of mechanically ventilated patients with pulmonary infection. Kaplan-Meier survival curves were used to analyze survival outcomes.Results:A total of 6 204 critically ill patients with pulmonary infection requiring mechanical ventilation were included, with 3 439 cases in propofol group and 2 765 cases in dexmedetomidine group. The baseline characteristics between the two groups were well balanced (standardized mean difference < 0.1) after IPTW adjustment. In the IPTW-adjusted cohort, the in-hospital all-cause mortality and 90-day all-cause mortality were significantly lower in dexmedetomidine group than in propofol group (439.2[18.7%] vs 563.6[24.1%], 618.0[26.3%] vs 733.6[31.3%], P<0.001), the results of Further Kaplan-Meier survival curve analysis showed that the 90-day all-cause mortality was significantly lower in dexmedetomidine group than in propofol group ( P<0.01). Conclusions:Compared with propofol, dexmedetomidine can decrease the mortality rate and improve the prognosis in mechanically ventilated patients with severe pulmonary infection when used for sedation.

Objective To investigate the regulatory effect and mechanism of Yiqi Jiedu Decoction(YQJD)on ionizing radiation-induced macrophage polarization and its correlation with the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB)signaling pathway.Methods Fifty-five specific-pathogen-free male Sprague-Dawley rats were randomly divided into blank(n=30),anduolin(n=10),and YQJD groups(n=15).They were respectively gavaged with deionized water,anduolin suspension(0.345 6 g/kg),and YQJD high-dose(20.88 g/kg)at a dose of 0.01 mL/g body weight once a day for seven consecutive days.2 hours after the last gavage,blood was collected from the abdominal aorta to prepare the control rat,andolin rat,and YQJD high-dose sera.Appropriate amounts of YQJD high-dose and control sera were mixed in a ratio of 1∶1 and 1∶3,respectively,to obtain YQJD medium-and low-dose rat serum.RAW264.7 cells were divided into blank(10%blank rat serum),model(10%blank rat serum),anduolin(10%anduolin rat serum),and YQJD-L,YQJD-M,YQJD-H groups(10%YQJD low-,medium-,and high-dose rat serum).Except for the blank group,the cells in other groups were irradiated with 12 Gy60 Co γ-rays once to establish the macrophage radiation injury model.At 24 h after irradiation,cell viability was detected using the CCK-8 method,and the cell migration rate was measured using the scratch test.Cell morphology was observed using phalloidin staining,tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)levels in the cell supernatant were quantified using enzyme-linked immunosorbent assay,and the proportion of M1 macrophages was detected using flow cytometry.TLR4,MyD88,and NF-κB protein expression were detected using Western blotting.Results Twenty-four hours after irradiation,compared with the blank group,the model group exhibited significantly reduced cell viability and migration rate(P<0.01),increased cell volume and pseudopodia formation,elevated TNF-α and IL-10 levels,an increased proportion of M1 macrophages,and upregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).Compared with the model group,each drug-treated group showed improved cell viability and migration rate(P<0.05,P<0.01),decreased cell volume,more regular cell shape,reduced TNF-α levels,lower M1-type macrophage proportion,and downregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).IL-10 level showed an upward trend.Conclusion YQJD can partially inhibit M1 macrophage polarization and suppress inflammatory responses,which may be related to the TLR4/MyD88/NF-κB signaling pathway.

Astragalus membranaceus possesses the function of enhancing immunity, protecting the liver, diuretic, anti-aging, anti-stress, anti-hypertensive, and more extensive antibacterial effects. Polysaccharides, one kind of the major active ingredients of A. membranaceus, are considered to be responsible for their versatile use. Now, a systematic summary of research progress and prospects of polysaccharides from A. membranaceus polysaccharides (AMPs) is necessary to facilitate their further study and application. In this review, the optimal extraction methods, structural features, biological activities, and applications of AMPs were emphasized. The structure-activity relationships are also analyzed and elucidated. Solvent, ultrasonic, microwave, enzyme-assisted, ultra-high pressure, and combined methods have been used to extract AMPs. Among them, solvent extraction is the most commonly used method because it is simple and easy to operate, but the efficiency needs to be improved further. The ultra-high pressure method is the most efficient but has a low economic return. AMPs exhibited various bioactivities, including immunomodulation, antitumor, and antidiabete. The structure-activity relationships revealed that different structure configurations, chain conformations, and physical properties would have different bioactivities. However, the new method for purification of certain polysaccharides, detailed structure-activity relationships (SAR), mechanisms of bioactivities, and quality control of AMPs need to be extensively investigated.

Objective To investigate the regulatory effect and mechanism of Yiqi Jiedu Decoction(YQJD)on ionizing radiation-induced macrophage polarization and its correlation with the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB)signaling pathway.Methods Fifty-five specific-pathogen-free male Sprague-Dawley rats were randomly divided into blank(n=30),anduolin(n=10),and YQJD groups(n=15).They were respectively gavaged with deionized water,anduolin suspension(0.345 6 g/kg),and YQJD high-dose(20.88 g/kg)at a dose of 0.01 mL/g body weight once a day for seven consecutive days.2 hours after the last gavage,blood was collected from the abdominal aorta to prepare the control rat,andolin rat,and YQJD high-dose sera.Appropriate amounts of YQJD high-dose and control sera were mixed in a ratio of 1∶1 and 1∶3,respectively,to obtain YQJD medium-and low-dose rat serum.RAW264.7 cells were divided into blank(10%blank rat serum),model(10%blank rat serum),anduolin(10%anduolin rat serum),and YQJD-L,YQJD-M,YQJD-H groups(10%YQJD low-,medium-,and high-dose rat serum).Except for the blank group,the cells in other groups were irradiated with 12 Gy60 Co γ-rays once to establish the macrophage radiation injury model.At 24 h after irradiation,cell viability was detected using the CCK-8 method,and the cell migration rate was measured using the scratch test.Cell morphology was observed using phalloidin staining,tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)levels in the cell supernatant were quantified using enzyme-linked immunosorbent assay,and the proportion of M1 macrophages was detected using flow cytometry.TLR4,MyD88,and NF-κB protein expression were detected using Western blotting.Results Twenty-four hours after irradiation,compared with the blank group,the model group exhibited significantly reduced cell viability and migration rate(P<0.01),increased cell volume and pseudopodia formation,elevated TNF-α and IL-10 levels,an increased proportion of M1 macrophages,and upregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).Compared with the model group,each drug-treated group showed improved cell viability and migration rate(P<0.05,P<0.01),decreased cell volume,more regular cell shape,reduced TNF-α levels,lower M1-type macrophage proportion,and downregulated TLR4,MyD88,and NF-κB protein expression(P<0.05,P<0.01).IL-10 level showed an upward trend.Conclusion YQJD can partially inhibit M1 macrophage polarization and suppress inflammatory responses,which may be related to the TLR4/MyD88/NF-κB signaling pathway.