ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveTo investigate the molecular mechanism of kaempferol-induced apoptosis in hepatic stellate cells （HSC） via the mitochondrial pathway. MethodsHSC were divided into five groups： blank control， transforming growth factor-β1（TGF-β1） （10 ng/mL） group， and groups treated with different concentrations of kaempferol （8， 16， 32 µmol/L）. Cell counting kit-8（CCK-8） assay was used to evaluate chrysin's effect on HSC proliferation. Transwell assay was used to assess chrysin's impact on HSC migration. Flow cytometry was used to detect apoptosis， reactive oxygen species （ROS） expression， and mitochondrial permeability transition pore （mPTP） opening across groups. 5，5′，6，6′-Tetrachloro-1，1′，3，3′-tetraethylimidacarbocyanine iodide （JC-1） assay was used to test mitochondrial membrane potential. Western blot analysis was performed to determine the expression levels of mitochondrial apoptosis-associated proteins， namely B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， Cleaved-Caspase3， Cleaved-Caspase9， and sirtuin 3 （SIRT3）. Transmission electron microscopy was used to examine mitochondrial structural alterations. Molecular docking and kinetic simulations were used to analyze the targeted interaction between kaempferol and NAD-dependent deacetylase sirtuin-3 （SIRT3）. ResultsCompared with the blank control group： in the TGF-β1 group， HSC proliferation and migration rates increased （P<0.01）， ROS levels and mPTP opening both increased （P<0.05）， the JC-1 red/green fluorescence ratio increased （P<0.01）， and the expression of Cleaved-Caspase-3， Cleaved-Caspase-9 and Bcl-2 increased （P<0.05）， whilst SIRT3 expression decreased （P<0.01）； the number of mitochondria increased （P<0.05）， and mitochondrial length decreased （P<0.01）. Compared with the TGF-β1 group： treatment with all groups of kaempferol reduced HSC proliferation and migration rates （P<0.01）， whilst simultaneously increasing the HSC apoptosis rate （P<0.01）； HSC ROS levels and mPTP opening both increased （P<0.05）； the JC-1 red/green fluorescence ratio decreased （P<0.01）， and the expression levels of Bax， Cleaved-Caspase-3 and Cleaved-Caspase-9 were upregulated （P<0.01）， whilst the expression levels of Bcl-2 and SIRT3 were downregulated （P<0.01）， and the number of mitochondria increased （P<0.05）； mitochondrial length was reduced （P<0.01）. Molecular docking and dynamics simulations revealed that the docking energy for kaempferol with SIRT3 was 108.89 kcal/mol； the RMSD curve stabilized after 20 ns； and RMSF analysis indicated that most residues exhibited minimal fluctuations. ConclusionKaempferol induces HSC apoptosis through the mitochondrial pathway， potentially via targeting SIRT3 and disrupting mitochondrial function.

Metabolic dysfunction-associated fatty liver disease （MAFLD） is currently the most prevalent chronic liver disease worldwide， and its disease spectrum encompasses simple steatosis， metabolic-associated steatohepatitis， hepatic fibrosis， liver cirrhosis， and hepatocellular carcinoma. Recent studies have shown that hepatic macrophages play an important regulatory role in the development and progression of MAFLD. Hepatic macrophages mainly consist of resident Kupffer cells and monocyte-derived macrophages， which participate in the pathophysiological processes of the disease by regulating inflammatory response， lipid metabolism， fibrogenic process， and cell death pathways. This article summarizes the diversity and functional plasticity of hepatic macrophages in MAFLD， reviews their interactions with hepatocytes and gut microbiota， elaborates on the core mechanism of hepatic macrophages and related signaling pathways in the progression of MAFLD， and further discusses novel therapeutic strategies targeting hepatic macrophages. An in-depth understanding of the mechanism of action of hepatic macrophages in MAFLD may provide a crucial theoretical basis for developing precise therapeutic approaches for this disease.

ObjectiveThis study explored the risk factors for malignant risks of pulmonary nodules based on clinical data，constructed an integrated traditional Chinese and Western medicine model for predicting malignant risks of pulmonary nodules， and visualized the prediction results by using a nomogram. MethodsBased on a cross-sectional survey study design，patients with pulmonary nodules who were hospitalized in the Department of Respiratory and Cardiothoracic Surgery of Guangdong Provincial Hospital of Traditional Chinese Medicine from April 2023 to January 2024 were included. The dataset was randomly divided into a training set and a validation set according to 7∶3. In the training set，predictive factors were selected through univariate Logistic regression analysis and Least Absolute Shrinkage and Selection Operator （LASSO） regression analysis，and Logistic regression models were built. The discriminative ability，calibration，and clinical decision-making curves of the Western medicine model and the integrated traditional Chinese and Western medicine prediction model were compared to select the optimal model，which was then visualized in a nomogram. ResultsThis study included a total of 366 patients，and they were divided into a training set （258 cases） and a validation set （108 cases）. Seven predictive factors were considered including age，preference for fatty and greasy foods，history of environmental or occupational exposure，Qi deficiency，Yang deficiency，nodule density，and nodule diameter. A Logistic regression model was constructed. A Western medicine model，defined as model1，was created using only age，history of environmental or occupational exposure，nodule density，and nodule diameter as predictive factors. In addition，an integrated traditional Chinese and Western medicine model，defined as model2，was created by adding preference for fatty and greasy foods， Qi deficiency，and Yang deficiency as predictive factors. Model2 demonstrated better predictive performance in both the training and validation sets. Its accuracy in training set was 0.740，with precision of 0.825， recall of 0.829， F1 score of 0.827， the area under the curve （AUC）of 0.865 （95% confidence interval （CI）：0.815-0.915）， and a Brier score of 0.122. The accuracy in validation set was 0.731， with precision of 0.776， recall of 0.831， F1 score of 0.803， AUC of 0.852 （95%CI：0.776-0.927）， and a Brier score of 0.149. The calibration curve and decision-making curve analysis showed that this model exhibited good consistency and clinical utility in prediction. The equation for the malignant probability of pulmonary nodules was defined as p=eY/(1+eY)，where Y=-4.187+0.022×（age）+1.079×（history of environmental or occupational exposure）+0.718×（preference for fatty and greasy foods）+ 1.403×（Qi deficiency）+0.732×（Yang deficiency）+0.349×（pure ground glass density nodules）+1.304×（partially solid density nodules）+0.148×（nodule diameter）. A nomogram was used for intuitive representation. ConclusionThis study develops a comprehensive assessment model for the malignant risk of pulmonary nodules， which integrates factors from traditional Chinese medicine and Western medicine. Embedding traditional Chinese medicine elements in the prediction model allows for a multidimensional assessment of relevant risk factors for nodule malignancy. This demonstrates good diagnostic performance and provides a reference for early identification of lung cancer.

Objective : To investigate the effects and underlying mechanisms of high mobility group nucleosome-binding domain protein 2(HMGN2) on lung adenocarcinoma cells. Methods : This work first analyzed the association between HMGN2 and lung adenocarcinoma tissues using The Cancer Genome Atlas(TCGA) database. Lung adenocarcinoma tissues and adjacent normal tissues were collected to compare the differential expression levels of HMGN2. The expression of HMGN2 mRNA in lung adenocarcinoma cell lines A549 and NC-H1299 were detected by qRT-PCR and Western blot. HMGN2 expression was knocked down using si-RNA technology, with the control group transfected with an equivalent amount of NC-siRNA, and the si-RNA group transfected with si-HMGN2. Stable transfected cell lines were established based on si-RNA knockdown efficiency. The effects of HMGN2 knockdown on the growth, movement, and spread of lung adenocarcinoma cells were assessed using CCK-8, Transwell assays, scratch assays, colony formation assays, and EdU assays. Transcriptome sequencing analysis revealed pathways related to tumorigenesis associated with HMGN2. The relative expression levels of MAPK pathway proteins after HMGN2 knockdown were detected by Western blot. Results : HMGN2 mRNA expression was significantly elevated in lung cancer tissues and lung adenocarcinoma cell lines(P<0.05). After HMGN2 knockdown, cell proliferation, migration, and invasion were significantly reduced(P<0.05), and the phosphorylation levels of the MAPK signaling pathway markedly decreased(P<0.05). Conclusion HMGN2 enhances the proliferation, migration, and invasion of lung adenocarcinoma cells, and its mechanism may be closely related to the activation of the MAPK signaling pathwayviaphosphorylation.

Objective: To investigate the mechanism of action of curcumol on mitochondrial autophagy in hepatic stellate cells and its molecular mechanism against liver fibrosis. Methods : Hepatic stellate cells were divided into blank group, model group(lipopolysaccharide 5 mg/L), and low, medium and high curcumol group(12.5, 25 and 50 mg/L); Thiazolyland(MTT) was used to detect the effects of curcumol on the viability of hepatic stellate cells; flow cytometry was used to detect the effects of curcumol on apoptosis of hepatic stellate cells; 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanine iodide(JC-1) was used to detect the mitochondrial membrane potential; effects of curcumol on mitochondrial morphology and autophagosome were detected by transmission electron microscopy; effect of curcumol on mitochondrial localisation were detected by fluorescent probe; Immunoblotting assay was performed to detect the effects of curcumin on PTEN-induced putative kinase 1(PINK1), Parkinson's disease protein(Parkin), microtubule-associated protein light chain 3(LC3), autophagy-associated protein(Beclin1), mitochondrial inner membrane translocase 23(Timm23), mitochondrial outer membrane translocase 20(Tomm20), Bcl-2 associated X protein(Bax), B lymphocytoma-2(Bcl2), cleaved-cysteine protease 3(Caspase3), α-smooth muscle actin(ɑ-SMA), collagen type Ⅰ(Collagen Ⅰ), and collagen type Ⅲ(Collagen Ⅲ) protein expression. Results : Compared with the blank control group, cell proliferation rate, Caspase3, Bcl2, LC3Ⅱ, Beclin1, PINK1, Parkin, ɑ-SMA, CollagenⅠ, CollagenⅢ proteins significantly increased in the model group(P<0.01), co-localisation of mitochondria and lysosomes increased, and the number of mitochondrial autophagosome significantly increased(P<0.01), while Timm23 and Tomm20 proteins, mitochondrial membrane potential decreased significantly(P<0.01), apoptosis rate decreased, and Bax protein expression decreased. Compared with the model group, after curcumol intervention, cell proliferation rate, Bcl2, Timm23, Tomm20, α-SMA, CollagenⅠ and CollagenⅢ protein expression significantly decreased in the curcumol low-, medium-and high-concentration groups(P<0.01), and the mitochondrial membrane potential significantly decreased(P<0.01), whereas apoptosis rate, Caspase3, Bax, LC3Ⅱ, Beclin1, PINK1 and Parkin proteins significantly increased(P<0.05), the co-localisation of mitochondria and lysosomes increased, and the number of mitochondrial autophagosome significantly increased(P<0.01). Conclusion Curcumol exerts ameliorative effects on hepatic fibrosis by modulating mitochondrial hyperautophagy mediated by the PINK1/Parkin signaling pathway, and promoting hepatic stellate cell apoptosis.

Lentiviral vectors(LVs)are key gene delivery tools for integrating target genes into the host genome,but they may also pose risks of insertional mutagenesis.The vector copy number(VCN)in cells is critical for determining the safety of gene modification.However,the reliability and accuracy of its quantification process are influenced by multiple factors.Developing cell reference materials with specific vector copy numbers represents a viable approach to enhance the reliability and consistency of measurement results,enabling quality control of the quantification process and traceability of outcomes.However,the preparation of such reference materials faces challenges in cell sample design,preparation protocols,and advanced quantification techniques.In this study,T lymphocyte cell line Jurkat-based reference materials with LV gene copy numbers of 1 and 2 copy/cell were developed.A high-precision duplex digital polymerase chain reaction(dPCR)method was established to quantify the LV gene and endogenous genes simultaneously.Additionally,the results of dPCR were cross-validated through next-generation sequencing and flow cytometric analysis.Ultimately,confocal microscopy characterization results showed that the developed cell reference materials had intact morphology.The quantification result of VCN-1 was(1.07±0.11)copy/cell,and that of VCN-2 was(2.09±0.21)copy/cell.These cell reference materials demonstrated compliance with stability and homogeneity requirements,and could be applied for quality control throughout the VCN measurement workflow and metrological traceability,improving the accuracy,comparability,and validity of copy number measurements.

Rheumatoid arthritis(RA)is a chronic autoimmune disease of unknown etiology that can cause joint destruction and deformity.As a small molecule cytokine,the chemokine C-X-C motif chemokine ligand 12(CXCL12)regulates the pathogenesis of rheumatoid arthritis by binding to the specific receptor CXC chemokine receptor 4(CXCR4).Therefore,based on the bio-logical characteristics of CXCL12 and CXCR4,this paper intro-duces the pathogenesis of CXCL12/CXCR4 in RA and summari-zes the progress in RA-related research,with the aim of providing clinical value for understanding the pathogenesis of RA and de-veloping novel therapeutic targets.

Objective:To investigate the effect of FOXQ1 expression on chemoresistance in colorectal cancer and analyze its regulatory role in SIRT1 expression and p53 deacetylation under DNA damage response(DDR)condi-tions.Methods:qRT-PCR was used to detect FOXQ1 mRNA expression levels in SW620 cells and SW620 cells stimulated with cisplatin(CDDP).Lentiviral vectors were constructed for FOXQ1 overexpression and RNA interference.The cells were divided into three groups:FOXQ1 overexpression group(oe-FOXQ1),FOXQ1 RNA interference group(sh-FOXQ1),and a control group transfected with an empty vector(NC).The half-maximal inhibitory concentration(IC50)of CDDP in each group was determined using the CCK-8 assay.Apoptosis level and cell viability were assessed using the Annexin V-APC/7-ADD apoptosis detection kit and Calcein/PI staining.Western blot analysis was performed to evaluate the effect of FOXQ1 on SIRT1 expression and acetylated p53 levels.The SIRT1 pathway inhibitor(S)-Selisi-stat was introduced to observe changes in p53 acetylation levels.Results:Compared to normal colon tissues,FOXQ1 expression was significantly upregulated in SW620 cells(P＜0.05),and low-dose CDDP stimulation further en-hanced its expression(P＜0.05).After 24 hours of CDDP treatment,the IC50 values for the oe-FOXQ1,sh-FOXQ1,and NC groups were 58.3 μmol/L,36.4 μmol/L,and 43.7 μmol/L,respectively,with statistically significant differences among the groups(P＜0.05).Compared to the NC group,the oe-FOXQ1 group showed a decrease in late apoptotic cell count(P＜0.05),while the sh-FOXQ1 group exhibited an increase(P＜0.05).Cytotoxic fluorescence staining re-vealed that the proportion of cell death was lower in the oe-FOXQ1 group and higher in the sh-FOXQ1 group com-pared to the NC group(P＜0.05).Protein expression analysis showed that FOXQ1 and SIRT1 levels were higher in the oe-FOXQ1 group and lower in the sh-FOXQ1 group compared to the NC group(P＜0.05).FOXQ1 overexpression pro-moted p53 deacetylation,while the addition of the SIRT1 pathway inhibitor(S)-Selisistat restored p53 acetylation levels(P＜0.05).Conclusion:FOXQ1 promotes p53 deacetylation by upregulating SIRT1 expression,thereby inhibiting DDR-induced apoptosis.