ObjectiveSpinal cord injury (SCI) directly impairs the regulatory function of the autonomic nervous system, induces intestinal dysfunction, and significantly reduces patients’ quality of life. Preclinical studies have shown that electroacupuncture (EA) therapy can regulate the brain-gut axis and is used to treat central nervous system diseases such as major depressive disorder, Alzheimer’s disease and Parkinson’s disease. Recent research has established that fecal microbiota transplantation (FMT) from EA-treated SCI rats restored intestinal motility and colonic morphology. However, it remains unclear whether the regulation of gut microbiota by EA therapy directly contributes to neural repair after SCI. This study aims to explore whether gut microbiota mediates the neuroprotective effect of EA in the treatment of SCI and its possible mechanism. MethodsThe study employed RNA transcriptome analysis of spinal cord tissue to characterize gene expression profiles and to identify key signaling pathways following EA treatment for SCI. Hematoxylin-Eosin (HE) staining and Nissl staining were used to observe the morphological changes in spinal cord tissue. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were applied to detect the effects of EA on the expression of proteins related to nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) -dependent pyroptosis. Using 16S rDNA sequencing, the study observed alterations in gut microbiota diversity and community composition in SCI rats. Prior to establishing SCI models, rats were pretreated with an antibiotic cocktail to induce gut dysbiosis, and the effects on intestinal function and spinal cord neural repair were evaluated. FMT was performed to investigate the regulatory effects of post-EA FMT on motor function, general status, liver and spleen indices, and NLRP3-mediated pyroptosis in SCI rats. ResultsEA improved motor function and reduced regulated neuronal cell death in SCI rats. Transcriptomic analysis demonstrated the activation of immune- and inflammation-related pathways post-SCI, including NOD-like receptors, nuclear factor-kappa B(NF-κB), and Toll-like receptor (TLR) pathways. EA primarily influenced intestinal inflammation and autoimmune functions. 16S rDNA sequencing illustrated that EA did not alter the diversity of gut microbiota. However, EA altered the gut microbiota composition in SCI rats, increasing Lactobacillus and Akkermansia genera while rebalancing the Firmicutes/Bacteroidetes ratio. Furthermore, depletion of gut microbiota by antibiotics disrupted the intestinal barrier, reduced the expression of intestinal barrier proteins Zonula Occludens-1 (ZO-1) and Occludin, elevated serum lipopolysaccharide-binding protein (LBP) levels, exacerbated spinal cord tissue damage, and hindered motor function recovery in SCI rats. FMT from donors treated with EA reduced LBP levels in the intestine, blood, and spinal cord of rats, inhibited the TLR4 myeloid differentiation primary response protein 88 (MyD88)-NF‑κB pathway and NLRP3-dependent pyroptosis, and improved motor function. On the other hand, FMT treatment resulted in decreased body weight and food intake, whereas FMT using EA-treated donors effectively alleviated these alterations. ConclusionEA effectively alleviated neuroinflammatory responses in rats with SCI, primarily through regulating the gut microbiota and suppressing the NLRP3-dependent pyroptosis signaling pathway.

ObjectiveSpinal cord injury (SCI) directly impairs the regulatory function of the autonomic nervous system, induces intestinal dysfunction, and significantly reduces patients’ quality of life. Preclinical studies have shown that electroacupuncture (EA) therapy can regulate the brain-gut axis and is used to treat central nervous system diseases such as major depressive disorder, Alzheimer’s disease and Parkinson’s disease. Recent research has established that fecal microbiota transplantation (FMT) from EA-treated SCI rats restored intestinal motility and colonic morphology. However, it remains unclear whether the regulation of gut microbiota by EA therapy directly contributes to neural repair after SCI. This study aims to explore whether gut microbiota mediates the neuroprotective effect of EA in the treatment of SCI and its possible mechanism. MethodsThe study employed RNA transcriptome analysis of spinal cord tissue to characterize gene expression profiles and to identify key signaling pathways following EA treatment for SCI. Hematoxylin-Eosin (HE) staining and Nissl staining were used to observe the morphological changes in spinal cord tissue. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were applied to detect the effects of EA on the expression of proteins related to nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) -dependent pyroptosis. Using 16S rDNA sequencing, the study observed alterations in gut microbiota diversity and community composition in SCI rats. Prior to establishing SCI models, rats were pretreated with an antibiotic cocktail to induce gut dysbiosis, and the effects on intestinal function and spinal cord neural repair were evaluated. FMT was performed to investigate the regulatory effects of post-EA FMT on motor function, general status, liver and spleen indices, and NLRP3-mediated pyroptosis in SCI rats. ResultsEA improved motor function and reduced regulated neuronal cell death in SCI rats. Transcriptomic analysis demonstrated the activation of immune- and inflammation-related pathways post-SCI, including NOD-like receptors, nuclear factor-kappa B(NF-κB), and Toll-like receptor (TLR) pathways. EA primarily influenced intestinal inflammation and autoimmune functions. 16S rDNA sequencing illustrated that EA did not alter the diversity of gut microbiota. However, EA altered the gut microbiota composition in SCI rats, increasing Lactobacillus and Akkermansia genera while rebalancing the Firmicutes/Bacteroidetes ratio. Furthermore, depletion of gut microbiota by antibiotics disrupted the intestinal barrier, reduced the expression of intestinal barrier proteins Zonula Occludens-1 (ZO-1) and Occludin, elevated serum lipopolysaccharide-binding protein (LBP) levels, exacerbated spinal cord tissue damage, and hindered motor function recovery in SCI rats. FMT from donors treated with EA reduced LBP levels in the intestine, blood, and spinal cord of rats, inhibited the TLR4 myeloid differentiation primary response protein 88 (MyD88)-NF‑κB pathway and NLRP3-dependent pyroptosis, and improved motor function. On the other hand, FMT treatment resulted in decreased body weight and food intake, whereas FMT using EA-treated donors effectively alleviated these alterations. ConclusionEA effectively alleviated neuroinflammatory responses in rats with SCI, primarily through regulating the gut microbiota and suppressing the NLRP3-dependent pyroptosis signaling pathway.

OBJECTIVE To investigate the apoptosis-inducing effect of esculetin （Esc） on acute myeloid leukemia （AML） HL-60 cells by regulating the protein kinase B （AKT）/S-phase kinase-associated protein 2 （SKP2）/MutT homolog 1 （MTH1） pathway. METHODS AML HL-60 cells were randomly divided into control group （routine culture）， Esc low-concentration group （L-Esc group， 25 μmol/L Esc）， Esc medium-concentration group （M-Esc group， 50 μmol/L Esc）， Esc high-concentration group （H-Esc group， 100 μmol/L Esc）， and high-concentration of Esc+ SC79 （AKT agonist） group （100 μmol/L Esc+5 μmol/L SC79）. Cell proliferation in each group was detected by MTT assay and colony formation assay. The level of reactive oxygen species （ROS） in cells was measured by using the CM-H2DCFDA fluorescent probe. Cell apoptosis was analyzed by flow cytometry. Western blot assay was performed to detect the expression levels of apoptosis-related proteins [B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved caspase-3]， AKT/SKP2/MTH1 pathway-related proteins （p-AKT， AKT， SKP2， MTH1）， along with the upstream and downstream proteins of AKT phosphatidylinositol 3-kinase （PI3K）， cyclin-dependent kinase inhibitor 1 （P21） and cyclin-dependent kinase inhibitor 1B （P27）. RESULTS Compared with control group， the cell viability， colony number， and the phosphorylation levels of AKT and PI3K proteins as well as protein expressions of SKP2， MTH1 and Bcl-2 were significantly decreased （P＜0.05）， while ROS level， apoptosis rate， and the expression levels of Bax， cleaved caspase-3， P21 and P27 proteins were significantly increased （P＜0.05）. Moreover， the effects of Esc exhibited concentration-dependence （P＜0.05）. Compared with H-Esc group， above indexes of high-concentration of Esc+ SC79 group were reversed significantly （P＜0.05）. CONCLUSIONS Esc may promote massive ROS production and induce activation of apoptosis in HL-60 cells by inhibiting the AKT/SKP2/MTH1 pathway， thus inhibiting the proliferation of HL-60 cells.

This study systematically examined the effects of atomization as a transdermal permeation enhancer on the transdermal delivery of active ingredients with different physicochemical properties and investigated the underlying mechanisms. Porcine ear skin served as an in vitro model, and skin permeation experiments were conducted using Franz diffusion cells. Receptor fluids were collected at specific time points, and samples of the stratum corneum and viable epidermis–dermis were separated and prepared. Quantitative and visualization methods, including high-performance liquid chromatography, fluorescence derivatization, and confocal laser scanning microscopy, were used to compare skin retention and transdermal permeation of adenosine (AD), tranexamic acid (TXA), calcein (CAL), and tocopheryl acetate (TA) under atomized and non-atomized conditions. Additionally, transepidermal water loss (TEWL) measurements and attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) were employed to analyze the mechanism behind the penetration enhancement caused by atomization. The findings showed that atomization increased skin retention within the stratum corneum and viable epidermis–dermis, as well as the transdermal permeation of all four active ingredients to varying degrees. The most significant enhancement was observed for TXA, which has a relatively low molecular weight and moderate lipophilicity, while the enhancement effects for highly hydrophilic AD and highly lipophilic TA were comparatively limited; however, atomization significantly increased the skin retention of TA. TEWL results indicated that skin permeability increased within 4 hours after atomization and largely recovered within 8 hours. ATR-FTIR analysis suggested that atomization may facilitate transdermal delivery by disrupting the lipid order and keratin conformation in the stratum corneum.This study provides theoretical support and experimental references for the advancement of atomization transdermal delivery technology, as well as the design and development of related atomization products.

Objective To investigate the effect of long non-coding RNA(lncRNA)potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1(KCNQ1OT1)on oxidative glucose deprivation/reoxygenation(OGD/R)-induced microglia injury through regulating the miR-145-5p/Rho-associated coiled-coil forming protein kinase 1(ROCK1)axis.Methods Microglia N9 were divided into the control group(normal culture),the OGD/R group(OGD/R-induced injury),the sh-NC group(transfected with sh-NC after OGD/R-induced injury),the sh-KCNQ1OT1 group(transfected with sh-KCNQ1OT1 after OGD/R-induced injury),the sh-KCNQ1OT1+inhibitor NC group(co-transfected with sh-KCNQ1OT1 and inhibitor NC after OGD/R-induced injury),and the sh-KCNQ1OT1+miR-145-5p inhibitor group(co-transfected with sh-KCNQ1OT1 and miR-145-5p inhibitor after OGD/R-induced injury).RT-qPCR was applied to detect the expression of lncRNA KCNQ1OT1,miR-145-5p,and ROCK1 mRNA in cells.The expression of ROCK1 protein was detected by Western blot.CCK-8 was applied to detect the cell proliferation.Flow cytometry was applied to detect cell apoptosis.ELISA was applied to detect the levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in cells.The action sites of miR-145-5p with lncRNA KCNQ1OT1 and ROCK1 were predicted by bioinformatics website,and their targeting relationships were verified by dual-luciferase reporter assay.Results Compared with the control group,the expression of lncRNA KCNQ1OT1,the expression of ROCK1 mRNA and protein,the apoptosis rate,and the levels of IL-6,TNF-α,and IL-1β in cells were all increased,and the level of miR-145-5p and cell survival rate were all decreased in the OGD/R group(P<0.05).Compared with the OGD/R group and the sh-NC group,the expression of lncRNA KCNQ1OT1,the expression of ROCK1 mRNA and protein,the apoptosis rate,and the levels of IL-6,TNF-α,and IL-1β in cells were all decreased,and the level of miR-145-5p and cell survival rate were all increased in the sh-KCNQ1OT1 group,with significant differences(P<0.05).Compared with the sh-KCNQ1OT1+inhibitor NC group,the expression of ROCK1 mRNA and protein,the apoptosis rate,and the levels of IL-6,TNF-α,and IL-1β in cells were all increased,and the expression of miR-145-5p and cell survival rate were all decreased in the sh-KCNQ1OT1+miR-145-5p inhibitor group,with significant differences(P<0.05).Bioinformatics website showed that miR-145-5p had targeted action sites with lncRNA KCNQ1OT1 and ROCK1,and dual-luciferase reporter assay confirmed that miR-145-5p had targeting relationships with lncRNA KCNQ1OT1 and ROCK1(P<0.05).Conclusion Silencing lncRNA KCNQ1OT1 can alleviate OGD/R-induced microglia injury via upregulating the expression of miR-145-5p and targeting the down-regulation of ROCK1 expression.

Aim To explore the action mechanism of isoquercitrin(IQ)in ameliorating anxiety based on network pharmacology,cellular transcriptomics,molecu-lar docking and animal experiments.Methods The common targets of anxiety disorders and IQ were ob-tained by using relevant databases.The protein-protein interaction network,the biological function and signa-ling pathway enrichment analysis were conducted by u-sing the common targets.Primary hippocampal neurons were cultured in vitro,and corticosterone was added to induce neurons to establish a corticosterone injury mod-el.IQ treatment was added to the culture system,and transcriptomics was used to screen for differentially ex-pressed genes and enrich for differentially expressed pathways.Subsequently,the results were validated by quantitative real-time polymerase chain reaction(qRT-PCR).Possible targets and signaling pathways for IQ treatment on anxiety were speculated and screened u-sing network pharmacology,transcriptomics and molec-ular docking.The anxiety rat model was constructed,and the anxiety state of rats was evaluated after IQ in-tervention,and the protein expression level of hippo-campus was detected to verify the relevant mechanism.Results Network pharmacology,cellular transcrip-tome,and molecular docking analyses revealed that the key mechanism of IQ for anxiety may be related to the BDKRB2/PI3K/Akt signaling pathway.Animal exper-iments showed that IQ was effective in improving anxie-ty behaviour and learning memory ability in rats.IQ increased the movement distance and residence time in the central area of the open field,the time and number percentage of entries into the open arm in the elevated plus maze,and the spontaneous alternations score in the Y maze in rats,and significantly elevated protein expression of BDKRB2,PI3K,Akt and decreased pro-tein expression of NF-κB in the hippocampus.Conclu-sions Isoquercitrin can effectively treat anxiety,and the mechanism of action may be related to the regula-tion of BDKRB2/PI3K/Akt signaling pathway in hip-pocampus.

AIM To investigate the effects of Xuesaitong Capsules(Panax notoginseng saponins)on ischemia/reperfusion injury in a mouse model of skin frostbite.METHODS The mice were randomly divided into the control group,the model group,the dexamethasone group(1 mg/kg),and the low-dose,medium-dose,and high-dose Xuesaitong Capsules groups(0.036,0.072,and 0.144 g/kg),with eight mice in each group.A frostbite model was established using a dry ice-cooled ceramic(ferrite)magnet.On the 2nd day after modeling,each group started its corresponding dosing by gavage for 14 consecutive days.The wound healing,histopathological changes,and serum levels of high-sensitivity C-reactive protein(hs-CRP),thromboxane B2(TXB2),6-keto-prostaglandin F1α(6-K-PGF1α),nitric oxide(NO)and endothelin(ET)were assessed using ELISA.The superoxide dismutase(SOD)activity and malondialdehyde(MDA)levels in skin tissues were measured biochemically.The protein expressions of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,Toll-like receptor(TLR)4 and phosphorylated nuclear factor-KB p65(p-NF-κB p65)in skin tissues were determined by Western blot.Additionally,LncRNA H19 mRNA expression in skin tissues was evaluated using RT-qPCR.RESULTS After the final administration,compared with the control group,the model group exhibited partial scab detachment,wound healing,and larger wound areas;hyperkeratosis with incomplete keratinization,detachment of the dermis and subcutaneous tissue,partial loss of appendages,subcutaneous edema,and dilated,congested,and hemorrhagic stromal vessels with extensive lymphocyte infiltration revealed by the histopathological examination;elevated serum levels of hs-CRP,TXB2,and ET(P＜0.05,P＜0.01);decreased 6-K-PGF1α and NO levels(P＜0.05,P＜0.01);reduced SOD activity in skin tissues(P＜0.01);increased MDA levels(P＜0.01);and upregulated protein expressions of TNF-α,IL-1β,IL-6,TLR4 and p-NF-κB p65,as well as LncRNA H19 mRNA expression(P＜0.05,P＜0.01).Compared with the model group,the group intervened with high-dose Xuesaitong Capsules displayed reduced wound areas(P＜0.01);decreased serum levels of hs-CRP,TXB2 and ET(P＜0.05,P＜0.01);increased 6-K-PGF1α and NO levels(P＜0.05,P＜0.01);enhanced SOD activity(P＜0.05,P＜0.01);reduced MDA level in skin tissues(P＜0.05,P＜0.01);and down-regulated TNF-α,IL-1β,IL-6,TLR4 and p-NF-κB p65 protein expressions and suppressed LncRNA H19 mRNA expression in skin tissues as well(P＜0.05,P＜0.01).CONCLUSION Xuesaitong Capsules alleviate ischemia/reperfusion injury in frostbite-injured mice by exerting anti-inflammatory and anti oxidative stress effects and restoring vascular endothelial function mediated by the downregulation of LncRNA H19 expression and inhibition of the TLR4/NF-κB signaling pathway.

Aim To explore the action mechanism of isoquercitrin(IQ)in ameliorating anxiety based on network pharmacology,cellular transcriptomics,molecu-lar docking and animal experiments.Methods The common targets of anxiety disorders and IQ were ob-tained by using relevant databases.The protein-protein interaction network,the biological function and signa-ling pathway enrichment analysis were conducted by u-sing the common targets.Primary hippocampal neurons were cultured in vitro,and corticosterone was added to induce neurons to establish a corticosterone injury mod-el.IQ treatment was added to the culture system,and transcriptomics was used to screen for differentially ex-pressed genes and enrich for differentially expressed pathways.Subsequently,the results were validated by quantitative real-time polymerase chain reaction(qRT-PCR).Possible targets and signaling pathways for IQ treatment on anxiety were speculated and screened u-sing network pharmacology,transcriptomics and molec-ular docking.The anxiety rat model was constructed,and the anxiety state of rats was evaluated after IQ in-tervention,and the protein expression level of hippo-campus was detected to verify the relevant mechanism.Results Network pharmacology,cellular transcrip-tome,and molecular docking analyses revealed that the key mechanism of IQ for anxiety may be related to the BDKRB2/PI3K/Akt signaling pathway.Animal exper-iments showed that IQ was effective in improving anxie-ty behaviour and learning memory ability in rats.IQ increased the movement distance and residence time in the central area of the open field,the time and number percentage of entries into the open arm in the elevated plus maze,and the spontaneous alternations score in the Y maze in rats,and significantly elevated protein expression of BDKRB2,PI3K,Akt and decreased pro-tein expression of NF-κB in the hippocampus.Conclu-sions Isoquercitrin can effectively treat anxiety,and the mechanism of action may be related to the regula-tion of BDKRB2/PI3K/Akt signaling pathway in hip-pocampus.

Objective To discuss the value of the multi-parameter imaging of dual-energy computed tomography(DECT)combined with apparent diffusion coefficient(ADC)value of brain metastases in predicting lung adenocarcinoma with epidermal growth factor receptor(EGFR)gene mutation.Methods The DECT and MRI parameters of 90 patients with lung adenocarcinoma and brain metastases were collected.DECT parameters included normalized iodine concentrations in the arterial and venous phases(N1CA,NICV)and slope of the energy spectrum curves in the arterial and venous phases(kA,kv).The MRI parameters of brain metastases included ADC and relative ADC(rADC)of solid area,necrotic area,and peritumoral edema.Predictive model were constructed for EGFR gene mutation according to the parameters above.Results NICV,kv,ADC and rADC of solid area and peritumoral edema in the EGFR positive group were higher than those in the EGFR negative group,which had statistical significances(P＜0.05).Model1 and model2 were established by combining DECT or MRI parameters,while model3 was established by combining DECT and MRI parameters,which had the best predictive efficiency for EGFR gene mutation,with an area under the curve(AUC)of 0.846.Conclusion The predictive model combining DECT parameters with ADC value of brain metastases can improve the prediction efficiency of EGFR gene mutation in advanced lung adenocarcinoma.

Objective To discuss the value of the multi-parameter imaging of dual-energy computed tomography(DECT)combined with apparent diffusion coefficient(ADC)value of brain metastases in predicting lung adenocarcinoma with epidermal growth factor receptor(EGFR)gene mutation.Methods The DECT and MRI parameters of 90 patients with lung adenocarcinoma and brain metastases were collected.DECT parameters included normalized iodine concentrations in the arterial and venous phases(N1CA,NICV)and slope of the energy spectrum curves in the arterial and venous phases(kA,kv).The MRI parameters of brain metastases included ADC and relative ADC(rADC)of solid area,necrotic area,and peritumoral edema.Predictive model were constructed for EGFR gene mutation according to the parameters above.Results NICV,kv,ADC and rADC of solid area and peritumoral edema in the EGFR positive group were higher than those in the EGFR negative group,which had statistical significances(P＜0.05).Model1 and model2 were established by combining DECT or MRI parameters,while model3 was established by combining DECT and MRI parameters,which had the best predictive efficiency for EGFR gene mutation,with an area under the curve(AUC)of 0.846.Conclusion The predictive model combining DECT parameters with ADC value of brain metastases can improve the prediction efficiency of EGFR gene mutation in advanced lung adenocarcinoma.