ObjectiveBased on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， network pharmacology and molecular docking techniques， to explore the pharmacodynamic material basis and mechanism of Renshen Wuweizi Tang in treating spleen-lung Qi deficiency syndrome. MethodsThe chemical components in the decoction of Renshen Wuweizi Tang were systematically characterized and identified by UPLC-Q-TOF-MS/MS， and network pharmacology was used to screen potential active ingredients， collect component targets and gene sets related to spleen-lung Qi deficiency syndrome， and obtain protein interaction relationships through STRING. Cytoscape 3.9.1 was used to construct a "formula-syndrome" association network and calculate topological feature values. Gene ontology（GO） function and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis were performed on core genes to explore potential pharmacodynamic links， the average shortest path between the formula-drug target network and the pharmacodynamic link gene network was calculated to discover dominant pharmacodynamic links， and MCODE plugin was used to identify core gene clusters from the dominant pharmacodynamic links， which were validated using Gene Expression Omnibus（GEO）， and molecular docking was performed between key components and core targets. ResultsOne hundred and thirty-seven components were identified in the negative ion mode， and eighty components were identified in the positive ion mode. After deduplication， a total of 185 components were identified， mainly composed of triterpenoid saponins（49） and flavonoids（54）. Based on the "formula-syndrome" correlation network analysis， energy metabolism was determined to be the dominant pharmacodynamic link of Renshen Wuweizi Tang in the treatment of spleen-lung Qi deficiency syndrome. The results of molecular docking showed that 7 components（adenosine， atractylenolide Ⅱ， atractylenolide Ⅲ， ginsenoside Rg₁， glycyrrhizin B₂， glycyrrhizin E₂ and campesterol） from 4 medicinal materials（Ginseng Radix et Rhizoma， Atractylodis Macrocephalae Rhizoma， Glycyrrhizae Radix et Rhizoma and Poria） in this formula might regulate energy metabolism by acting on 6 targets， namely cyclic adenosine monophosphate-response element binding protein 1（CREB1）， glyceraldehyde-3-phosphate dehydrogenase（GAPDH）， interleukin（IL）-6， nuclear transcription factor（NF）-κB1， peroxisome proliferator-activated receptor α（PPARα）， and tumor necrosis factor（TNF）， thus improving the symptoms of diseases related to spleen-lung Qi deficiency syndrome. ConclusionThis study established a UPLC-Q-TOF-MS/MS for rapid characterization and identification of chemical components in the decoction of Renshen Wuweizi Tang， expanding the understanding of the material composition of this formula， and found that 7 components might act on the key advantageous pharmacodynamic link "energy metabolism" through 6 targets to improve the related symptoms of spleen-lung Qi deficiency syndrome. This can provide a reference for the subsequent exploration of the material benchmark and mechanism of the famous classical formula.

Objective: To explore the relationship between visual acuity and physical fitness of university freshmen, so as to provide reference for myopia prevention and control for freshmen. Methods: From October to November 2022, 2 160 college freshman without majoring in public safety administration, selected from Guangxi Police College in 2022 by using the stratified cluster random sampling method, were reviewed for the results of visual acuity test and physical fitness scores. The physical fitness indices were evaluated by using the Z scores of physical fitness test scores, and the strength of association between the level of physical fitness index and myopia was analyzed by using Logistic regression model. Results: Among 2 160 college freshman without majoring in public safety administration, 917 (42.5%) students were diagnosed screening myopia, including 66 (3.1%) cases of high myopia, 383 (17.7%) cases of moderate myopia and 468 (21.7%) cases of mild myopia. The differences in the distribution of visual acuity tests among students with different physical fitness indices, body mass index, and gender were statistically significant ( Z/H=54.50, 49.53, 15.51, P <0.01). Low level and low middle level physical fitness indices were associated with screening myopia among freshmen[ OR (95% CI )=2.81(1.93-4.08),1.87(1.38-2.54)], and low level physical fitness indexes were associated with high myopia [ OR (95% CI )=7.22(2.33-22.32)] ( P <0.01). Conclusions Screening myopia among college freshman without majoring in public safety administration is related to physical fitness, and low level and low middle level physical fitness index are risk factors for myopia. Improving the level of physical fitness might be effective in preventing myopia.

ObjectiveTo compare the hepatic bile acid profile between a mouse model of metabolic-associated steatohepatitis （MASH） induced by a high-fat， high-sugar， and high-cholesterol diet combined with intraperitoneal injection of 10% carbon tetrachloride （CCl₄） and MASH cases in clinical practice， and to investigate the feasibility of this model in studying drug interventions on bile acid profile in MASH. MethodsA total of 30 male C57BL/6J mice were randomly divided into control group and model group， with 15 mice in each group. The mice in the control group were given normal diet and drinking water and weekly injections of olive oil， and those in the model group were given a high-fat， high-sugar， and high-cholesterol diet， high-sugar drinking water， and weekly injections of CCl₄+olive oil. At the end of weeks 8， 12， and 16， 5 mice were selected from each group to collect samples. Behavioral assessments were performed， and body weight and liver wet weight were measured； liver pathology and lipid deposition were evaluated by HE staining， SAF scoring， oil Red O staining， the semi-quantitative analysis of stained area， the serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST）， and liver triglyceride （TG） content； Sirius Red staining was performed for liver tissue to assess liver fibrosis； ultra-performance liquid chromatography-tandem mass spectrometry and targeted metabolomics were used to measure the hepatic bile acid profile， including cholic acid （CA）， glycocholic acid （GCA）， chenodeoxycholic acid （CDCA）， glycochenodeoxycholic acid （GCDCA）， ursodeoxycholic acid （UDCA）， tauroursodeoxycholic acid （TUDCA）， hyodeoxycholic acid （HDCA）， and glycodeoxycholic acid （GDCA）. The independent-samples t test was used for comparison of normally distributed continuous data between two groups， and the Wilcoxon rank-sum test was used for comparison of non-normally distributed continuous data between two groups. ResultsCompared with the control group at the same time point， the model group had disheveled and dull fur， reduced activity， and relatively slow reactions at weeks 8， 12， and 16， as well as significant increases in liver wet weight （P<0.05）， the serum level of ALT （P<0.05）， the content of TG in the liver （P<0.05）， and SAF score （P<0.05）. As for the differentially expressed bile acids in liver tissue， compared with the control group at week 8， the model group had significantly higher levels of CA and CDCA and significantly lower levels of UDCA， TUDCA， HDCA， and GDCA （all P<0.05）； compared with the control group at week 12， the model group had significantly higher levels of CA， GCA， CDCA， and GCDCA and significantly lower levels of UDCA and HDCA （all P<0.05）； compared with the control group at week 16， the model group had significantly higher levels of CA， GCA， CDCA， GCDCA， and TUDCA and significantly lower levels of UDCA， HDCA， and GDCA （all P<0.05）. As for the differentially expressed bile acids in the bile acid pool of liver tissue， compared with the control group at week 8， the model group had significantly higher levels of CA and CDCA and significantly lower levels of UDCA， TUDCA， GDCA， and HDCA （all P<0.05）； compared with the control group at weeks 12 and 16， the model group had significantly higher levels of GCA and GCDCA and significantly lower levels of UDCA， GDCA， and HDCA （all P<0.05）. ConclusionThere are significant changes in the hepatic bile acid profile in a mouse model of MASH induced by a high-fat， high-sugar， and high-cholesterol diet combined with CCl₄， which are similar to the changes in bile acids in MASH cases in clinical practice， suggesting that this model can be used to explore the interventional effect of drugs on the bile acid profile in MASH.

Aim To establish a stable hepatic stellate cell ( HSC ) -specific G protein-coupled receptor kinase 2 ( GRK2 ) knockout mice and provide the important animal model for further studying the biological function of GRK2 in HSC. Methods The loxP-labeled Grk2 gene mouse (Grk2

Aim To investigate the therapeutic effect of the MW-9 on ulcerative colitis(UC)and reveal the underlying mechanism, so as to provide a scientific guidance for the MW-9 treatment of UC. Methods The model of lipopolysaccharide(LPS)-stimulated RAW264.7 macrophage cells was established. The effect of MW-9 on RAW264.7 cells viability was detected by MTT assay. The levels of nitric oxide(NO)in RAW264.7 macrophages were measured by Griess assay. Cell supernatants and serum levels of inflammatory cytokines containing IL-6, TNF-α and IL-1β were determined by ELISA kits. Dextran sulfate sodium(DSS)-induced UC model in mice was established and body weight of mice in each group was measured. The histopathological damage degree of colonic tissue was assessed by HE staining. The protein expression of p-p38, p-ERK1/2 and p-JNK was detected by Western blot. Results MW-9 intervention significantly inhibited NO release in RAW264.7 macrophages with IC50 of 20.47 mg·L-1 and decreased the overproduction of inflammatory factors IL-6, IL-1β and TNF-α(P<0.05). MW-9 had no cytotoxicity at the concentrations below 6 mg·L-1. After MW-9 treatment, mouse body weight was gradually reduced, and the serum IL-6, IL-1β and TNF-α levels were significantly down-regulated. Compared with the model group, MW-9 significantly decreased the expression of p-p38 and p-ERK1/2 protein. Conclusions MW-9 has significant anti-inflammatory activities both in vitro and in vivo, and its underlying mechanism for the treatment of UC may be associated with the inhibition of MAPK signaling pathway.

Objective To observe the therapeutic effects and mechanisms of Guanyuan Mingmen Sequential Acupuncture on rats with premature ovarian insufficiency(POI)model.Methods Female SD rats were divided into the blank group,the model group,the protein kinase A(PKA)inhibitor(H89)+acupuncture group,and the acupuncture group,with 12 rats in each group.Except for the blank group,the POI model was prepared by gavage with Tripterygium Glycosides Tablets in the other three groups of rats.After the model was successfully established,the blank group and the model group were bundled once a day;in the acupuncture group,Guanyuan(RN4)point was taken during the intermotility period,and in the pre-motility period,Mingmen(DU4)point was taken;in the H89+acupuncture group,the intervention was performed in accordance with the acupuncture protocol of the acupuncture group,and H89 was injected intraperitoneally for 30 minutes prior to each acupuncture session.Continuous intervention was performed for 20 days.Samples were taken from each group of rats in the first estrus period and in proestrus period after intervention.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of follicle stimulating hormone(FSH)and estradiol(E2)during the estrous phase,Western Blot was used to measure the protein expressions of follicle stimulating hormone receptor(FSHR)and aromatase P450(P450arom)during the estrous phase,and the activity of granulocytes during the estrous phase and the proestrus phase were measured using the cell-counting kit 8(CCK-8)method.The immunohistochemistry method was used to detect the protein expression of pre-motility proliferating cell nuclear antigen(PCNA).Results(1)Compared with the blank group,the serum FSH level of the model group and H89+acupuncture group was significantly increased(P＜0.01),and the E2 level was significantly decreased(P＜0.001);there was no difference between the FSH level of the H89+acupuncture group and that of the model group(P＞0.05),and the E2 level of the H89+acupuncture group was lower than that of the model group(P＜0.05);the FSH level of the acupuncture group was lower than that of the model group and that of the H89+acupuncture group(P＜0.05),had no difference with the blank group(P＞0.05),E2 level was significantly higher than the model group and H89+ acupuncture group(P＜0.01),still being lower than the blank group(P＜0.05).(2)The protein expressions of FSHR and P450arom in the model group and H89 + acupuncture group was lower than those in the blank group;the protein expression of FSHR in the H89 + acupuncture group was not different from that in the model group(P＞0.05),while the protein expression level of P450arom was lower than that in the model group(P＜0.05);the protein expression levels of FSHR and P450arom in the acupuncture group were higher than those in the model group and H89 + acupuncture group,but still lower than those in the blank group(P＜0.05).(3)Both GCs activity and average optical density value of PCNA in the model group and H89+acupuncture group were lower than the blank group(P＜0.05);both GCs activity and average optical density value of PCNA in the H89+acupuncture group were lower than the model group(P＜0.05);the activity of GCs and average optical density value of PCNA of the acupuncture group were significantly higher than that of the model group and H89+acupuncture group(P＜0.05 or P＜0.01).Conclusion Guanyuan Mingmen Sequential Acupuncture can regulate sex hormone levels,increase GCs activity and promote GCs cell proliferation by up-regulating protein expressions of follicle stimulating hormone receptor(FSHR)/cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)pathway FSHR,P450arom,thus improving POI.

Objective To investigate the effects of metformin(Met)on the proliferation of pancreatic cancer cells under different glucose concentration culture conditions,and to find the potential role of miR-139-5p in the process.Methods PANC-1 cells were treated with different concentrations of metformin(0/5/10/20 mmol/L)in 25 mmol/L(high-glucose group,HG)or 5 mmol/L(normal-glucose group,NG)glucose culture,cell proliferation,apoptosis,migration and cell cycle were detected after 48 h.The expression of miR-139-5p was quantitatively detected by RT-qPCR,and the miR-139-5p mimics were transfected into PANC-1 cells to clarify the role of miR-139-5p.Results Metformin inhibited the proliferation,promoted apoptosis,and induced S phase and G2/M phase arrest of PANC-1 cells under in high glucose and normal glucose culture conditions,and its anti-proliferation and pro-apoptosis effects were more significant in the normal glucose groups.The expression of miR-139-5p was up-regu-lated by metformin treatment in normal but not in high glucose culture.Further studies showed that miR-139-5p mimics inhibited of PANC-1 cells proliferation without metformin pre-incubation and enhanced the anti-prolifera-tion effect of 5 mmol/L metformin.The pro-apoptotic effect of 10 mmol/L metformin in normal glucose culture conditions.Conclusions In normal-glucose culture conditions,metformin can inhibit proliferation,induce apop-tosis and cell cycle arrest of PANC-1 cells more significantly than in higher-glucose culture,which may be partly related to the up-regulation of miR-139-5p.

ObjectiveThe purpose of this study was to investigate the effects of transcutaneous electrical acupoint stimulation (TEAS) on cognitive function of vascular dementia (VD) rats and its mechanism. MethodsVD rat model was established by modified two-vessel occlusion (2-VO). After modeling, TEAS and electroacupuncture (EA) were used to stimulate Baihui and Zusanli points of rats respectively for 14 d. After treatment, novel object recognition test, Morris water maze test, and Y maze test were used to evaluate the spatial memory and learning ability of rats. Hematoxylin and eosin staining was used to observe the morphology of hippocampal neurons. Transmission electron microscopy was used to observe the ultrastructure of hippocampal mitochondria. Enzyme-linked immunosorbent assay kits were used to detected the levels of SOD, CAT, GSH-Px, MDA and ROS in serum of rats. Western blot was used to detect the expression of PGC-1α, TFAM, HO-1, NQO1 proteins in the hippocampus, Keap1 protein in the cytoplasm and Nrf2, NRF1 proteins in the nucleus. ResultsAfter treatment for 14 d, compared to the model group, the escape latency of VD rats decreased, while the discrimination index, the times of rats crossing the original platform area, the residence time in the original platform quadrant, and the percentage of alternation increased. TEAS can improve the structure of hippocampal neurons and mitochondria of VD rats, showing that neurons were arranged more regularly and distributed more evenly, nuclear membrane and nucleoli were clearer, and mitochondrial swelling were reduced, mitochondrial matrix density were increased, and mitochondrial cristae were more obvious. The levels of SOD, GSH-Px and CAT in serum increased significantly, while the concentration of MDA and ROS decreased. TEAS also up-regulated the expression levels of PGC-1α TFAM, NQO1 and HO-1 proteins in the hippocampus and Nrf2, NRF1 proteins in the nucleus, but down-regulated the Keap1 protein in the cytoplasm. ConclusionTEAS can improve cognition, hippocampal neurons and mitochondrial structure of VD rats, and the effect is better than EA. The mechanism may be the activation of PGC-1α mediated mitochondrial biogenesis and antioxidant stress, which also provides a potential therapeutic technology and experimental basis for the treatment of VD.

Objectives:To find the impact of outpatient security in health seeking behavior,health protection and medical ex-pense for chronic diseases.Methods:Based on the hypertension patients'all medical visits records of urban and rural residents who were insured under the basic medical insurance system in sample region of Nanjing from 2019 to 2021,the quantitative relationship between chronic disease outpatient treatment and patients'healthcare behaviors,health outputs,and healthcare costs was analyzed by an individual-time two-way fixed effect model and Utest test.Results:When the annual outpatient reimbursement ratio is in the range of(42.99％,64.11％),strengthening the security for outpatient service could make people seek medical advice reasonably and achieve a better health outcome,as well as controlling the medical expenses.Conclusion:Properly raising actual compensation for chronic outpatient care could lead to the rational health-seeking behaviors,safeguarding health for the insured,and on this basis,achieve control of medical costs and effective use of the basic medical insurance pool.

Objective:To analyze the methylation characteristics of the lymphocyte-specific protein-tyrosine kinase (LCK) promoter region in the peripheral blood circulation of rheumatoid arthritis (RA) patients and its correlation with clinical indicators.Methods:Targeted methylation sequencing was used to compare the methylation levels of 7 CpG sites in the LCK promoter region in the peripheral blood of RA patients with healthy controls (HC) and osteoarthritis (OA) patients. Correlation analysis and ROC curve construction were performed with clinical information.Results:Non-parametric tests revealed that compared with HC [0.53(0.50, 0.57)] and OA patients [0.59(0.54, 0.62), H=47.17, P<0.001], RA patients [0.63(0.59, 0.68)] exhibited an overall increase in methylation levels. Simultaneously, when compared with the HC group [0.38(0.35, 0.41), 0.59(0.55, 0.63), 0.60(0.55, 0.64), 0.59(0.55, 0.63), 0.58(0.53, 0.62), 0.45(0.43, 0.49), 0.57(0.54, 0.61)], the RA group [0.46(0.42, 0.49), 0.70(0.65, 0.75), 0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] showed a significant elevation in methylation levels at CpG sites cg05350315_60, cg05350315_80, cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-5.63, -5.89, -5.91, -5.89, -5.98, -5.95, -5.95, all P<0.001). Compared with the OA group [0.65(0.59, 0.69), 0.65(0.60, 0.69), 0.64(0.58, 0.68), 0.50(0.45, 0.54), 0.63(0.58, 0.67)], the RA group [0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] exhibited a significant increase in methylation levels at CpG sites cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-3.56, -3.52, -3.60, -3.67, -3.62; P=0.036, 0.042, 0.031, 0.030, 0.030). Furthermore, Pearson correlation coefficient analysis revealed a positive correlation between the overall methylation level in this region and C-reactive protein (CRP) ( r=0.19, P=0.004) and erythrocyte sedimentation rate ( r=0.14, P=0.035). The overall methylation level of the LCK promoter region in the CRP (low) group [0.63 (0.58, 0.68)] was higher than that in the CRP (high) group [0.65(0.61, 0.70)], with statistically significant differences ( Z=2.60, P=0.009). Finally, by constru-cting a ROC curve, the discriminatory efficacy of peripheral blood LCK promoter region methylation levels for identifying RA patients, especially seronegative RA patients, from HC and OA groups was validated, with an AUC value of 0.78 (95% CI: 0.63, 0.93). Conclusion:This study provides insights into the methylation status and methylation haplotype patterns of the LCK promoter region in the peripheral blood of RA patients. The overall methylation level in this region is positively correlated with the level of inflammation and can be used to differentiate seronegative RA patients from the HC and OA patients.