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”.

Objective To establish and test a 3-year prognosis model for elderly intertrochanteric fractures after intramedullary nail fixation.Methods A total of 205 elderly patients with intertrochoteric fracture of femur who underwent intramedullary nail fixation in our hospital from April 2019 to April 2021 were selected for observation study,followed up for 3 years after surgery,according to the Harris score,they were divided into the good group and the poor group.Univariate analysis was conducted to investigate the influencing factors of disease prognosis in the two groups 3 years after surgery.After the variables were screened by LASSO regression and cross-validation method,the independent influencing factors of the 3-year postoperative prognosis were analyzed by multi-factor Logistic regression,and the prediction model of the nomogram was built,and the model was evaluated and validated.Results Among 205 elderly patients with intertrochanteric fractures of the femur who underwent intramedullary nailing fixation,201 were followed up for 3 years.Among them,148 patients had a Harris score of ≥70 points,while 53 patients had a score of＜70 points.Univariate analysis results showed that the poor group had a higher proportion of patients with age,stroke,osteoporosis,Evans-Jensen type Ⅲ and Ⅳ fractures,poor intraoperative reduction,and a tip-apex distance of ≥ 30 mm compared to the good group,and had a lower lateral wall thickness than the good group,the difference was statistically significant(P＜0.05).LASSO regression analysis and cross-validation were used to screen variables.Multivariate Logistic regression analysis showed that stroke(OR=2.127,95％CI:1.478-3.061)and fracture Evans-Jensen classification Ⅲ(OR=1.149,95％CI:1.105-1.195)and type Ⅳ(OR=1.187,95％CI:1.143-1.233),intraoperative reduction was not good(OR=3.290,95％CI:2.319-4.668),apex distance ≥ 30 mm(OR=1.413,95％CI:1.066-1.874)was an independent associated risk factor for disease outcome 3 years after surgery,the external wall thickness(OR=0.600,95％CI:0.428-0.841)was an independent correlated risk factor for 3-year prognosis(P＜0.05).Based on the results of multiple factors,a nomogram prediction model was drawn,and the results showed that the model had certain predictive value for the disease outcome three years after surgery.The evaluation and verification results showed that when the threshold probability was 0-96％,the model had good clinical applicability and positive clinical net benefit.Conclusion Stroke,fracture Evans-Jensen classification Ⅲ and Ⅳ,poor intraoperative reduction,apical distance ≥30 mm,and lateral wall thickness are independent and relevant factors for 3-year prognosis of senile intertrochanteric fractures after intramedullary nail fixation.The establishment of a nematographic model has good predictive value for postoperative long-term prognosis and positive clinical net benefit.It can be used as an effective model to predict the long-term prognosis of postoperative disease.

Objective:To observe the effect of acupuncture synchronized exercise therapy on sensory and motor functions of lower limbs in stroke patients,and to provide scientific basis for the optimization of rehabilitation program for stroke patients.Method:Seventy stroke patients hospitalized in the Rehabilitation Department of the Affiliated Hospital of Shan-dong University of Traditional Chinese Medicine from March 2022 to July 2022 were selected and randomly di-vided into synchronous group and control group,35 patients in each group.The patients in the synchronous group received routine exercise therapy and synchronized acupuncture treatment,while the patients in the con-trol group received acupuncture and subsequent exercise therapy continuously.Before and after treatment,the revised Nottingham sensory assessment scale(reNSA),Fugl-Meyer sensory rating scale,Fugl-Meyer motor function assessment(FMA),Berg rating scale,modified Barthel index rating scale(MBI)and modified Ash-worth spasm rating scale(MAS)were used to evaluate the curative effects.Result:After treatment,the scores of reNSA deep sensation,reNSA compound sensation and Fugl sensation in two groups were significantly increased(P<0.05).The scores of deep and shallow sensation in the synchro-nous group were significantly higher than those in the control group,while there was no significant difference between the two groups in the scores of compound sensation and Fugl sensation(P>0.05).After treatment,the FMA-UE score,Berg score and MBI score in the two groups were significantly increased(P<0.05),and the FMA-UE score,Berg score and MBI score of the synchronous group were also significantly higher than those of the control group(P<0.05).The composition of MAS scores in both groups also improved significant-ly after treatment,but there was no significant difference between the two groups before or after treatment(χ2=2.469,P=0.650;χ2=1.651,P=0.648).Conclusion:Acupuncture synchronized exercise therapy can effectively improve the sensory and motor functions of the lower limbs after stroke,and its effect is better than that of exercise therapy and acupuncture sequen-tial therapy.

Objective To observe the effect of acupuncture synchronized with speech training on speech function of patients with post-stroke motor aphasia.Methods Sixty inpatients with post-stroke motor aphasia were selected from the Affiliated Hospital of Shandong Univer-sity of Traditional Chinese Medicine,from January to August,2023.They were randomly divided into control group(n=30)and synchronous group(n=30).Both groups received acupuncture and speech training;the con-trol group received acupuncture in the morning and speech training in the afternoon,while the synchronous group received acupuncture and speech training synchronously,for three weeks.They were assessed with Chi-nese Rehabilitation Research Center Standard Aphasia Examination(CRRCAE),Non-Language-Based Cognitive Assessment(NLCA)and Communication Activities of Daily Living(CADL)before and after treatment.Results The subscores of CRRCAE and NLCA,and score of CADL increased in both groups(|t|>2.081,P<0.05)after treatment,and they were better in the synchronous group than in the control group(|t|>2.680,P<0.05).Conclusion Synchronous mode of acupuncture and speech training is more effective on post-stroke motor aphasia than time-sequence mode.

Objective To observe the effect of synchronous acupuncture and articulation training on spastic dysarthria after stroke.Methods From January to August,2023,64 stroke patients in the Affiliated Hospital of Shandong University of Tradi-tional Chinese Medicine were selected,and randomly divided into control group(n=32)and synchronous group(n=32).Both groups received routine neurological treatment and basic articulation training.The control group added asynchronous acupuncture,while the synchronous group added synchronous acupuncture,for four weeks.Before and after treatment,the modified Frenchay Dysarthria Assessment(m-FDA),Speech Intelligibility Test(SIT),maximum phonation time(MPT)and maximum counting ability(MCA)were used to evaluate the curative effects.Results After treatment,the scores of m-FDA in all the dimensions decreased in both groups(t>2.882,P<0.01);ex-cept for the jaw dimension,the scores of m-FDA in reflexes,respiration,lips,soft palate,larynx,tongue and speech dimensions were significantly lower in the synchronous group than in the control group(t>2.050,P<0.05).After treatment,the results of SIT,and MPT and MCA significantly increased in both groups(t>21.061,P<0.001),and they were better in the synchronous group than in the control group(t>11.412,P<0.001).Conclusion Synchronous acupuncture and articulation training can effectively alleviate the severity of spastic dysarthria after stroke,which was superior to asynchronous acupuncture and articulation training.

Kupffer cells(KC),the hepatic resident macrophages,are located in the sinusoids,and play a crucial role in clearing intestinal-derived pathogens and damaged red blood cells,regulating iron and fat metabolism,and maintaining immune toler-ance.In acute and chronic hepatopathy,KC are implicated in liver injury and repair by crosstalk between other hepatocellular popula-tions such as hepatocytes,endothelial cells,hepatic stellate cells and so on,by which maintain liver homeostasis.KC are highly plas-tic,and different liver disease models and different hepatic microenvironmental signals can induce changes in the phenotype and func-tion of hepatic macrophages.In this review,we summarize the subsets of KC and their functions in specific liver disease models based on cell origin,cell polarization,cell surface markers,and gene expression profiles identified by single-cell sequencing techniques.We are aiming to target specific KC subsets to prevent or treat related liver diseases,and promote the progress of precision medicine for liver diseases.

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 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.