ObjectiveTo investigate the mechanism by which adrenoceptor alpha 2A (Adra2a) regulates lipopolysaccharide (LPS)-induced inflammation in primary hepatocytes from lipopolysaccharide-binding protein (LBP) knockout mice (Lbp^-/-). MethodsPrimary hepatocytes from C57BL/6J and Lbp^-/- mice were isolated using a two-step perfusion method. An in vitro inflammatory model was established by LPS stimulation, and an in vivo inflammatory mouse model was established by intraperitoneal injection of LPS. The in vitro experiments were grouped as follows: Control group, LPS group, BRL+LPS group, OE-NC+LPS group, and OE-Adra2a+LPS group. The Control group served as the blank control. The LPS group involved stimulating primary hepatocytes with LPS. The BRL+LPS group involved pretreating primary hepatocytes with BRL-44408 maleate followed by LPS stimulation. The OE-NC+LPS group involved transfecting primary hepatocytes with an empty vector followed by LPS stimulation. The OE-Adra2a+LPS group involved transfecting primary hepatocytes with a lentivirus overexpressing Adra2a, followed by LPS stimulation. The in vivo experimental groups were divided into Control', LPS', BRL+LPS', OE-NC+LPS', and OE-Adra2a+LPS' groups. The Control' group served as the blank control. The LPS' group received intraperitoneal injection of LPS. The BRL+LPS' group received intraperitoneal injection of BRL-44408 maleate for pretreatment, followed by LPS injection. The OE-NC+LPS' group received intraperitoneal injection of empty vector for pretreatment, followed by LPS injection. The OE-Adra2a+LPS' group received intraperitoneal injection of a lentivirus overexpressing Adra2a for pretreatment, followed by LPS injection. Cell viability after Adra2a inhibition and overexpression was assessed via the Cell Counting Kit-8 (CCK-8) assay. RT-qPCR measured changes in gene expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) after Adra2a inhibition and overexpression. Western blotting was performed to detect Adra2a protein expression and phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase (JNK) following LPS stimulation. ResultsIn vitro experiments revealed that LPS stimulation significantly decreased Adra2a protein expression in primary hepatocytes from C57BL/6J mice compared to the Control group (P<0.05), whereas it increased in primary hepatocytes from Lbp^-/- mice (P<0.001). Compared to the LPS group, the BRL+LPS group exhibited significantly increased cell viability (P<0.01), reduced TNF-α, IL-6, and IL-1β gene transcription levels (P<0.01, P<0.001, P<0.001), and decreased phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK (P<0.01, P<0.001, P<0.001). Compared with the OE-NC+LPS group, the OE-Adra2a+LPS group showed significantly decreased cell viability (P<0.001), increased gene transcription levels of TNF-α, IL-6, and IL-1β genes (P<0.001, P<0.01, P<0.001), and elevated phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK (P<0.001, P<0.01, P<0.001). In vivo experiments showed that, compared with the LPS' group, the BRL+LPS' group exhibited significantly reduced phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK (P<0.001, P<0.01, P<0.01). In the OE-Adra2a+LPS' group, the phosphorylation levels of ERK1/2, p38, and JNK were significantly elevated compared to the OE-NC+LPS' group (P<0.01, P<0.001, P<0.01). ConclusionLPS stimulation can cause a significant increase in Adra2a protein expression in primary hepatocytes of Lbp^-/- mice. Adra2a protein can regulate the level of LPS-induced inflammation in primary hepatocytes of Lbp^-/- mice through the MAPK signaling pathway.

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 anti‑pancreatic fibrosis mechanism of Klotho‑derived peptide 7 （KL7） by observing its effect on a mouse model of chronic pancreatitis （CP） induced by cerulean， and to provide a basis for clinical medication. MethodsA total of 40 male BALB/c mice were randomly divided into control group， model group， low-dose KL7 group （2 mg/kg）， and high-dose KL7 group （4 mg/kg）， with 10 mice in each group. All mice except those in the control group were given intraperitoneal injection of cerulean （50 μg/kg） 6 times a day at an interval of 1 hour， twice a week for 4 consecutive weeks to establish a model of CP. The mice in the low-dose KL7 group and the high-dose KL7 group were treated with different doses of KL7 once a day for 4 consecutive weeks. In vivo imaging was used to observe the accumulation of KL7 in the pancreas； molecular docking was used to detect the binding of KL7 to transforming growth factor-β type Ⅱ receptor （TβRⅡ）； the mice were measured in terms of body weight and pancreatic weight； HE staining was used to observe the pathological changes of pancreatic tissue； Masson staining was used to observe the degree of pancreatic fibrosis； immunohistochemical staining was used to measure the expression of α-smooth muscle actin （α-SMA） and type Ⅰ collagen （COL1A1）； Western blotting was used to measure the protein expression levels of α-SMA， TβRII， and phosphorylated small mothers against decapentaplegic homolog 2/3 （p-Smad2/3） in pancreatic tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test and the Dunnett’s-T3 test were used for further comparison between two groups. ResultsKL7 was significantly enriched in the pancreatic tissue of CP mice， and there was a strong binding activity between KL7 and TβRⅡ. Compared with the control group， the model group had significant reductions in pancreatic mass and relative pancreatic mass （P<0.000 1）， with disordered structure of pancreatic tissue， an increase in inflammatory cell infiltration， and significant increases in fibrosis degree， the positive areas of α-SMA and COL1A1 （P<0.000 1）， and the protein expression levels of α-SMA， TβRⅡ， and p-Smad2/3 （P<0.05）. Compared with the model group， the high-dose KL7 group had significant increases in pancreatic mass and relative pancreatic mass （P<0.01）， with alleviation of structural damage of pancreatic tissue and inflammatory cell infiltration， a significant reduction in fibrosis degree， and significant reductions in the positive areas of α-SMA and COL1A1 （P<0.001） and the protein expression levels of α-SMA， TβRⅡ， and p-Smad2/3 （P<0.01）. ConclusionKL7 has a significant targeted therapeutic effect on pancreatic fibrosis in CP mice through specific binding of KL7 to TβRⅡ， thereby inhibiting the activation of the TGF-β/Smad signaling pathway.

Based on the discussion of "eight weak areas" in The Inner Canon of Yellow Emperor （《黄帝内经》）， combined with the typical rash manifestations of atopic dermatitis， it is believed that atopic dermatitis is mostly deficiency-excess complex， and that pathogens intruding eight weak areas are the core of its pathogenesis. The external cause is exterior deficiencies， with heat， wind， dampness and other pathogenic qi attacking. The heart， lungs， kidneys out of balance， and excess pathogen are the internal cause， in which fire constraint and excessive heat are the basis of the disease， the wind invading leads to the progress of the changes， dampness obstructing channels and colla-terals make the condition persistent. Internal and external pathogens combination and retention result to the course of the disease lingering and difficult to cure. The internal treatment is to regulate zang-fu organs， and the formula could use self-prescribed modified Qingrun Tongluo Decoction （清润通络汤）， clearing heart and reducing fire in order to clear the heat and cool the blood， moistening lungs and generating metal to consolidate the exterior and dispel the wind， and nourishing kidneys and draining water to dispel the dampness and activate the collaterals. The external treatment applies maceration， fire acupuncture， wrapping to dredge the eight weak areas and regulate qi and blood in channel， so as to expel pathogens.

Objective:To explore impacts of nuciferine(Nuci)on liver fibrosis and inflammatory reaction in cirrhotic rats through p38MAPK-PPARγ/NF-κB signal pathway.Methods:Six SD rats were randomly selected as control(NC)group,other rats were injected with thioacetamide(TAA)into peritoneum at a dose of 200 mg/kg to induce cirrhosis.Rats successfully modeled were randomly grouped into Model group,Nuci group(20 mg/kg Nuci),anisomycin group(5 mg/kg Nuci p38MAPK-PPARγ/NF-κB signal pathway activator anisomycin),and Nuci+anisomycin group(20 mg/kg Nuci+5 mg/kg anisomycin),with 6 rats in each group.The NC group and the Model group were given the same amount of normal saline once a day for 8 weeks.Serum inflammatory index and liver function index were detected by ELISA kits;HE staining was applied to detect pathological changes of liver;Masson staining was applied to detect liver fibrosis;Western blot was applied to detect pathway related proteins levels.Results:In NC group,cell structure was normal,only a small amount of collagen was deposited,no collagen fiber proliferation was observed,and liver lobule was clearly stained.Compared with NC group,hepatocytes in Model group were enlarged,accompanied by inflammatory cell infiltration,and in-creased collagen fiber proliferation,IL-1β,TNF-α,alanine aminotransferase(ALT),IL-6,hyaluronic acid(HA),aspartate amino-transferase(AST),type Ⅲ procollagen(PCⅢ),total bilirubin(TBIL)content,laminin(LN),collagen volume fraction,p-p38MAPK/p38MAPK,p-NF-κB p65/NF-κB p65 proteins levels were significantly increased(P<0.05),PPARγ protein level was significantly decreased(P<0.05).Nuci treatment improved inflammatory cell infiltration and necrosis in liver tissue,levels of IL-1β,IL-6,TNF-α,ALT,AST,TBIL,HA,LN,PCⅢ,collagen volume fraction,p-p38MAPK/p38MAPK,p-NF-κB p65/NF-κB p65 pro-teins were significantly decreased(P<0.05),PPARγ protein level was significantly increased(P<0.05),trend of anisomycin group was opposite to that of Nuci group;anisomycin eliminated therapeutic effect of Nuci on cirrhotic rats.Conclusion:Nuci may reduce liver fibrosis and inflammation in cirrhotic rats by down-regulating p38MAPK-PPARγ/NF-κB signal pathway.

Objective To investigate the role of cholinergic receptor muscarinic 3(Chrm3)in regulating lipopolysaccharide(LPS)-induced inflammation in peritoneal macrophages in lipopolysaccharide binding protein(LBP)-knockout(Lbp-/-)mice.Methods Peritoneal macrophages were isolated from wild-type and Lbp-/-mice to establish an LPS-induced inflammation model.Chrm3 expression in Lbp-/-mouse peritoneal macrophages was inhibited by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide(4-damp)and small interfering(siRNA)and Chrm3 overexpression was achieved by lentivirus transfection.For 4-damp inhibition,cells were divided into control,LPS,and inhibitor groups,and for siRNA transfection,cells were divided into control,LPS,si-normal control group,and si-Chrm3 groups.For overexpression,cells were divided into control,LPS,negative control,and overexpression groups.Changes in Chrm3 in response to LPS stimulation were verified by Western blot.The effects of 4-damp,si-Chrm3,and lentivirus on cell inflammation and survival were confirmed by Cell Counting Kit-8,quantitative polymerase chain reaction,and Western blot assays.Results Chrm3 protein expression was significantly elevated in Lbp-/-peritoneal macrophages post-LPS stimulation(P＜0.001),whereas there was no notable change in wild-type cells.The cell survival rate was significantly increased in the 4-damp and si-Chrm3 groups(P＜0.05,P＜0.01),and cell survival was significantly reduced in the overexpression group(P＜0.01).Furthermore,4-damp and si-Chrm3 significantly reduced expression levels of the inflammatory factors tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6(P＜0.01,P＜0.001),and phospho-extracellular signal-regulated kinase(p-ERK)(P＜0.01,P＜0.001),which are associated with cell damage and inflammation.In contrast,TNF-α,IL-1β,IL-6(P＜0.001),and p-ERK protein(P＜0.001)were significantly elevated in the overexpression group.Conclusions LPS stimulation upregulated the expression of Chrm3 and proinflammatory cytokines in Lbp-/-peritoneal macrophages.Specific downregulation of Chrm3 by 4-damp and si-Chrm3 significantly decreased LPS-induced proinflammatory cytokines in Lbp-/-peritoneal macrophages,while upregulation of Chrm3 using overexpressing lentivirus significantly elevated the expression of related inflammatory factors.Chrm3 is implicated in the regulation of the LPS-induced inflammation response in peritoneal macrophages in Lbp-/-mice.

Parkinson's disease(PD)is one of the most common neurodegenerative disorders.Recent evidence implicates pyroptosis as one of the pathogenic mechanisms in central nervous system disorders,although its specific mechanisms remain unclear.In this study,SH-SY5Y cells were transfected with py-roptosis-related proteins GSDME full-length(GSDME-F)or GSDME-N terminal(GSDME-N)plasmids revealed that GSDME-N significantly reduced mitochondrial membrane potential(P＜0.0001).To inves-tigate the mechanism by which GSDME mediates mitochondrial dysfunction,Western blotting analysis demonstrated that transfection with GSDME-N plasmids significantly increased BAX expression and en-hanced its translocation to mitochondria in both HEK 293T and SH-SY5Y cells(P＜0.05).SH-SY5Y cells treated with varying concentrations of rotenone(ROT)exhibited GSDME cleavage,elevated BAX expression(P＜0.05),increased mitochondrial BAX aggregation(P＜0.05),and reduced mitochondrial membrane potential(P＜0.01),as confirmed by Western blotting and JC-1 staining.Concurrently,MTT assays assessing cell viability and lactate dehydrogenase(LDH)release assays indicated that ROT in-duced these processes prior to pyroptosis.Furthermore,in a ROT-induced mouse PD model,ROT trig-gered GSDME cleavage,enhanced BAX expression,caused dopaminergic neuronal damage,and induced motor deficits.In summary,this study demonstrates that GSDME-N exacerbates mitochondrial damage and increases cytotoxicity by upregulating BAX expression and facilitating its mitochondrial translocation.This study provides novel insights into the role of GSDME in PD pathogenesis and suggests potential avenues for therapeutic intervention.

Objective:To investigate the clinical manifestations，etiology/triggers，treatment，and prognosis of children with cerebral venous sinus thrombosis（CVST）.Methods:A retrospective analysis was conducted on 36 children with CVST hospitalized at the Affiliated Children's Hospital of Xiangya School of Medicine,Central South University（Hunan Children's Hospital）from May 2014 to January 2024.A centralized telephone follow-up was performed in May 2024，and clinical data including symptoms，imaging findings，treatments，and outcomes were collected.According to the prognosis,the children were divided into favorable-prognosis group and poor-prognosis group,and the differences of clinical characteristics between the two groups were compared.The univariate Logistic regression was applied to identify factors associated with prognosis.Results:Among the 36 cases,there were 29 males and 7 females,ranging in age from 1 month to 13 years and 3 months,with a median age of 4.6(1.0,8.3) years.The common clinical manifestations included headache（24/25，96.0%），consciousness disorder（25/36，69.4%），vomiting（22/36，61.1%），seizures（14/36，38.9%），limb dysfunction（11/36，30.6%）.The leading etiologies were infection（14/36，38.9%），head trauma（8/36，22.2%），and tumors/chemotherapy（6/36，16.7%）.All 36 children underwent MRI+MRV examination of the head,and all of them had different degrees of CVST,the most commonly involved site was transverse sinus (28/36,77.8%).The favorable-prognosis group（ n=18）included 16 patients receiving anticoagulation and 2 trauma cases without anticoagulation.The poor-prognosis group（ n=18）comprised 9 anticoagulated and 9 non-anticoagulated patients.There were no significant differences in age,sex,clinical manifestations,etiology/inducement and thrombus site between the two groups ( P>0.05).However,the proportion of anticoagulant therapy in the favorable-prognosis group was higher than that in the poor-prognosis group(88.9% vs 50.0%).Among the 25 children receiving anticoagulant therapy,16 had a good prognosis (64.0%),while among the 11 children receiving no anticoagulant therapy,only 2 had a good prognosis (18.2%).The prognosis of children receiving anticoagulant therapy was better than that of those receiving no anticoagulant therapy.The difference was statistically significant ( P<0.05).Fourteen children were admitted with intracranial hemorrhage,with 8 receiving anticoagulant therapy (7 with good prognosis,accounting for 87.5%) and 6 not receiving anticoagulant therapy (only 1 with good prognosis,accounting for 12.5%).The prognosis of children receiving anticoagulant therapy was better than that of those receiving no anticoagulant therapy,and the difference was statistically significant ( P=0.026),with no increasing in intracranial hemorrhage after anticoagulant therapy.Univariate Logistic regression analysis showed that inducement/etiology,intracranial hemorrhage before treatment and prognosis were not related（ P >0.05），but anticoagulation treatment was associated with favorable outcomes（ OR=0.125，95% CI 0.017-0.614, P=0.009）. Conclusion:Infection is the primary etiology of pediatric CVST，with headache，lethargy，and vomiting as key symptoms.Transverse sinus is the most commonly involved site.Children suspected of CVST should be examined by MRI/MRV as soon as possible,and early anticoagulation therapy should be given after a clear diagnosis,so as to improve the prognosis.

Objective:To explore the expression of lamin (LMN) gene in Colorectal cancer (CRC) , as well as the effects of knockdown LMNA expression in colorectal cancer cells on its migration ability and related molecular mechanisms.Methods:Paraffin-embedded specimens of tumor tissues and corresponding peritumoral tissues were collected from 37 colorectal cancer patients for the detection of LMNA protein expression. Colorectal cancer cell line SW480 was cultured in vitro and divided into Mock group (transfected MOCk-siRNA) and LMNA group (transfected LMNA-siRNA) . Real-time quantitative PCR was used to detect the LMNA mRNA content in SW480 cells of experimental group and control group. The expression levels of LMNA, Wnt and β-catenin in SW480 cells of experimental group and control group were detected by Western blotting. The migration ability of cells in each group was detected by cell scratch test. The migration ability of cells in each group were detected by transwell assay.Results:Immunohistochemical test showed that the positive rate of LMNA protein in colorectal cancer tissues was 89.19% (33/37 cases) , and the expression rate in corresponding paracancer tissues was 48.65% (18/37 cases) . The expression level of LMNA in colorectal cancer tissues was significantly higher than that in paracancer tissues ( P<0.001) . siRNA decreased the expression of LMNA protein in colorectal cancer cells SW480. The scratch healing rate was (53.71±5.34) % in the experimental group and (83.84±6.98) % in the control group. The results of Transwell experiment showed that the number of successfully migrated cells in the experimental group was 34.92±5.11, and that in the control group was 93.87±12.57. The results showed that the migration ability of SW480 cells was significantly decreased after low expression of LMNA ( P<0.01) . Western blot results showed that the relative expression level of Wnt and β-catenin in LMNA group was 0.42±0.12 and 0.22±0.11 respectively. The relative expression levels of Wnt and β-catenin in MOCK group were 1.28±0.26 and 1.14±0.21 respectively. The expression levels of P16 and Wnt and β-catenin in PC3 cells with low LMNA expression were increased ( P<0.05) , while the expressions of WNT and β-catenin were decreased (both P<0.05) . Conclusion:The expression of LMNA was significantly increased in colorectal cancer, which may be related to the malignant degree of colorectal cancer. LMNA proteins may affect the migration ability of colorectal cancer cells by regulating the Wnt/β-catenin signaling pathway.