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.

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.

The radiation-free and unmarked body surface monitoring technology is developed for reducing the additional radiation dose generated by positioning error verification during radiotherapy positioning,and further reducing the positioning error and monitoring the displacement deviation of patients during radiotherapy in real time.At present,the widely used optical surface guided radiotherapy technology is also a type of radiotherapy guided by body surface monitoring.The system mainly uses optical imaging equipment as a tool to complete body surface scanning,three-dimensional reconstruction,real-time monitoring,etc.,thereby assisting doctors to carry out radiotherapy more accurately.Herein the study elaborates on the methods,technologies and research results of guided radiotherapy from the aspects of body surface markers,three-dimensional surface imaging systems and mobile devices,and provides prospects for future researches.

AIM To explore the clinical effects of Qijing Buzhong Yishen Decoction on patients with diabetic nephropathy due to Spleen-Kidney Qi Deficiency and Blood Stasis Obstructing Collaterals.METHODS One hundred and two patients were randomly assigned into control group(51 cases)for 3-month intervention of conventional treatment,and observation group(51 cases)for 3-month intervention of both Qijing Buzhong Yishen Decoction and conventional treatment.The changes in clinical effects,blood glucose indices(fasting blood glucose,2 h postprandial blood glucose,HbA1c,TIR),blood lipid indices(TC,TG,LDL-C),renal function indices(UACR,eGFR,24 h urinary albumin excretion rate,sustained urinary albumin excretion rate),inflammatory factors(IL-6,hs-CRP,TNF-α),TCM syndrome scores and incidence of adverse reactions were detected.RESULTS The observation group demonstrated higher total effective rate than the control group(P＜0.05).After the treatment,the two groups displayed decreased fasting blood glucose,2 h postprandial blood glucose,HbA1c,UACR,24 h urinary albumin excretion rate,sustained urinary albumin excretion rate,inflammatory factors,TCM syndrome scores(P＜0.05),and increased TIR,eGFR(P＜0.05),especially for the observation group(P＜0.05).No serious adverse reactions were observable in the two groups.CONCLUSION For the patients with diabetic nephropathy due to Spleen-Kidney Qi Deficiency and Blood Stasis Obstructing Collaterals,Qijing Buzhong Yishen Decoction can safely and effectively regulate the blood sugar levels,and improve renal functions.

Objective To develop a Knowledge-Attitude-Practice Questionnaire for Nursing Assistants on Humanistic Care and test its reliability and validity,in order to provide a valid tool for evaluating their humanistic care competence.Methods Based on the theory of knowledge,attitude and practice,the first draft of the questionnaire was developed through literature review,semi-structured interviews,2 rounds of Delphi expert consultation,and pre-surveys.All nursing assistants from 4 tertiary hospitals in Hubei Province were investigated by convenient sample,and the reliability and validity were tested from May to July,2024.Results 412 questionnaires were distributed and 399 valid questionnaires were recovered,with a valid questionnaire recovery rate of 96.84％.The final knowledge-attitude-practice questionnaire include 3 dimensions of knowledge,attitude and practice,with a total of 24 items and cumulative contribution rate was 65.338％.The CVI of each item in the questionnaire ranged from 0.800 to 1.000,with an average of 0.981.The confirmatory factor analysis showed that the scale model had a good fit.The total Cronbach's α coefficient of the questionnaire was 0.941,and the test-retest reliability coefficient of the questionnaire after 2 weeks was 0.923.Conclusion The knowledge-attitude-practice questionnaire has good reliability and validity,and it can be used as a valid tool for evaluating the competence of humanistic care of nursing assistants.

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.