BACKGROUND:Previous studies have found that neohesperidin can delay bone loss in ovariectomized mice and has the potential to treat osteoporosis,but its specific mechanism of action remains to be explored. OBJECTIVE:To explore the key targets and possible mechanisms of neohesperidin in the treatment of osteoporosis based on bioinformatics and cell experiments in vitro. METHODS:The gene expression dataset related to osteoporosis was obtained from GEO database,and the differentially expressed genes were screened and analyzed in R language.The osteoporosis-related targets were screened from GeneCards and DisGeNET databases,and the neohesperidin-related targets were screened from ChEMBL and PubChem databases,and the common targets were obtained by intersection of the three.The String database was used to construct the PPI network of intersection genes,and the key targets were screened.The DAVID database was used for GO and KEGG enrichment analysis.The AutoDock software was used to verify the molecular docking between the neohesperidin and the target protein.The effect of neohesperidin on osteogenic differentiation of C57 mouse bone marrow mesenchymal stem cells was detected.Complete medium was used as blank control group;osteogenic induction medium was used as the control group;and osteogenic induction medium containing different concentrations of neohesperidin(25,50 μmol/L)was used as experimental group.The expression of alkaline phosphatase,the degree of mineralization,the expression of osteogenic-related genes and target genes during osteogenic differentiation of cells were measured at corresponding time points. RESULTS AND CONCLUSION:(1)9 253 differentially expressed genes,2 161 osteoporosis-related targets,and 326 neohesperidin-related targets were screened.There were 53 common targets among the three.All 53 genes were up-regulated in osteoporosis samples.The PPI network screened the target gene PRKACA of research significance.GO function and KEGG pathway enrichment analysis showed that neohesperidin's treatment of osteoporosis through PRKACA target mainly depended on biological processes such as protein phosphorylation and protein autophosphorylation,acting on endocrine resistance,proteoglycan in cancer,and estrogen signaling pathway to play a therapeutic role.Molecular docking results showed that neohesperidin had a certain binding ability to the protein corresponding to the target PRKACA.(2)The results of alkaline phosphatase staining showed that neohesperidin could promote the expression of alkaline phosphatase in the early stage of osteogenic differentiation of mesenchymal stem cells.Alizarin red staining showed that neohesperidin could promote the mineralization of osteogenic differentiation of mesenchymal stem cells.RT-qPCR results showed that neohesperidin could increase the mRNA expression of alkaline phosphatase,PRKACA,and osteocalcin.(3)These results indicate that neohesperidin may promote osteogenic differentiation through PRKACA target on the estrogen signaling pathway to prevent and treat osteoporosis.

BACKGROUND:As a degradable scaffold material for bone tissue engineering,lactic acid is widely used in tissue regeneration and repair research,and plays an important role in promoting tissue healing,new bone formation and angiogenesis. OBJECTIVE:To observe the effect of lactic acid degradation products on osteoclasts and to investigate the effects of lactic-interfered osteoclast conditioned medium on the proliferation,migration and tube-forming capacity of human umbilical vein endothelial cells. METHODS:(1)The mouse monocyte macrophage cell line RAW264.7 at logarithmic growth period was selected,and adherent cells were cultured in the osteoclast induction medium(DMEM medium with nuclear factor-κB receptor-activating factor ligand and 10%fetal bovine serum)containing different concentrations of lactic acid(0,5,10,20 mmol/L).After 5 days of culture,tartrate-resistant acid phosphatase staining and cytoskeletal fibrillar actin staining were conducted.After 24 hours of culture,RT-PCR was used to detect the mRNA expression of tartrate-resistant acid phosphatase 5.(2)RAW264.7 cells at logarithmic growth period were selected and adherent cells were divided into two groups.Control group was cultured in the osteoclast induction medium,while experimental group was cultured in the osteoclast induction medium containing 10 mmol/L lactic acid.After 5 days of culture,the medium in each group was removed and the cells in the two groups were cultured in the serum-free DMEM medium for another 24 hours.Cell supernatant was then collected and used as the conditioned medium after mixed with an equal volume of DMEM medium containing 10%fetal bovine serum.Human umbilical vein endothelial cells at the logarithmic growth phase were taken and separately co-cultured with the conditioned medium of the control and experimental groups.The proliferation,migration and tube-forming ability of human umbilical vein endothelial cells were observed by cell counting kit-8 assay,migration assay,scratch assay and tube-forming assay.The mRNA and protein expression of angiogenesis-related genes and proteins were observed by RT-PCR and western blot. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining and cytoskeletal fibrillar actin staining showed that 5 and 10 mmol/L lactic acid promoted osteoclastic differentiation of RAW264.7 cells and the promoting effect of 10 mmol/L lactate was more significant.RT-PCR results showed that the expression of tartrate-resistant acid phosphatase-5 mRNA of osteoclast-related genes was the highest when the lactic acid concentration was 5,10,and 20 mmol/L(P<0.05),especially 10 mmol/L.Compared with the control group,the proliferation,migration and tube-forming abilities of human umbilical vein endothelial cells were significantly increased in the experimental group(P<0.05).Compared with the control group,the expression levels of vascular endothelial growth factor and angiogenin 1 mRNA and protein were increased in the experimental group(P<0.05).To conclude,lactate-induced osteoclast conditioned medium could promote the angiogenesis of endothelial cells,and the mechanism may be related to the promotion of the expression of vascular endothelial growth factor and angiogenin 1.

BACKGROUND:Traditional Chinese medicine has rich experience and unique advantages in the empirical treatment of phlegm-heat and Fu-organs excess syndrome of ischemic stroke.In order to further explore the therapeutic targets and mechanisms of traditional Chinese medicine for this disease,it is crucial to establish a stable and reliable animal model of phlegm-heat and Fu-organs excess syndrome combined with empirical symptoms of ischemic stroke. OBJECTIVE:To explore the establishment method and evaluation system of the rat model of ischemic stroke with phlegm-heat and Fu-organ excess syndrome. METHODS:Sixty male Sprague-Dawley rats were randomly divided into four groups:blank control group(n=12),ischemic stroke group(n=18),disease+syndrome group(n=18),phlegm-heat and Fu-organ excess syndrome group(n=12),all of which were given high-fat diet for 25 days.On the 26th day,the rats in the blank control group and ischemic stroke group were intragastrically given normal saline and high fat diet,while those in the other two groups were intragastrically given autologous feces suspension and high fat diet for 3 continuous days.After gavage,ischemic stroke models were established using the suture method in the ischemic stroke group and disease+syndrome group.The changes in diet,water intake,body mass,body temperature,fecal traits,nasal secretions,sputum in the throat,and tongue image were recorded.Neurological deficits,tongue image,blood lipid levels,morphological changes of brain tissue and carotid artery,and the serum levels of motilin and somatostatin were detected. RESULTS AND CONCLUSION:Compared with the control group,the rats in the disease+syndrome group had shortness of breath,listlessness,irritability,bradykinesia,a large number of secretions around the nose,audible and heavy sputum in the throat,decreased diet and water intake,increased body mass,body temperature,and slingual vein score,decreased fecal pellet count,Bristol score and fecal moisture content,increased serum total cholesterol,triglyceride,low-density lipoprotein and somatostatin levels,decreased motilin level,increased neurological deficit score,significant pathological changes of the carotid artery,and significant morphological changes of the brain tissue.The ischemic stroke group only showed pathological changes of ischemic brain tissue,without the characteristics of phlegm-heat and Fu-organ excess syndrome.The phlegm-heat and Fu-organ excess syndrome group could present with the typical characteristics of traditional Chinese medicine syndromes,without the pathological changes of brain tissue with ischemic stroke.To conclude,the compound modeling method of high-fat induction combined with suture method and autologous feces gavage can establish an animal model of ischemic stroke with phlegm-heat and Fu-organ excess syndrome.

BACKGROUND:The diabetic microenvironment can cause excessive M1 polarization of macrophages,and this hyperglycemic inflammatory state can inhibit osteogenic differentiation of bone marrow mesenchymal stem cells,thus affecting the healing of diabetic bone defects.Studies have indicated that mogroside V possesses anti-inflammatory,antioxidant,and hypoglycemic properties.However,its potential to modulate M1 polarization of macrophages and osteogenic differentiation of bone marrow mesenchymal stem cells under high glucose and inflammatory condition remains unclear. OBJECTIVE:To explore the effect of mogroside V on regulating M1 macrophage polarization and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells under high glucose and inflammatory condition. METHODS:Murine diabetic models were established using C57BL/6 mice.Bone marrow-derived macrophages were isolated from tibia and fibula of normal and diabetic mice,and cultured in low-glucose and high-glucose media.Then M1 polarization of bone marrow-derived macrophages was induced using lipopolysaccharide and interferon-γ.Bone marrow-derived macrophages were treated with 160,320,and 640 μmol/L mogroside V.Flow cytometry was employed to determine the proportion of F4/80+CD86+cells.qRT-PCR was utilized to assess mRNA expression levels of inducible nitric oxide synthase,interleukin 1β,and interleukin 6.ELISA was employed to evaluate tumor necrosis factor-α secretion in bone marrow-derived macrophage supernatants.Bone marrow mesenchymal stem cells were isolated from tibia and fibula of C57BL/6 suckling mice,and induced osteogenic differentiation using low-or high-glucose osteogenic induction medium.Bone marrow mesenchymal stem cells were treated with M1 macrophage-conditioned mediums with or without 320 μmol/L mogroside V in osteogenic differentiation process.qRT-PCR was employed to assess the mRNA expression of alkaline phosphatase,Runt-related factor 2,osteocalcin,and osteopontin on day 14 after osteogenic induction.Alizarin red staining and quantitative analysis were conducted to evaluate calcium deposition on day 21 after osteogenic induction. RESULTS AND CONCLUSION:(1)Flow cytometry results showed that with the treatment of 320 and 640 μmol/L mogroside V,the proportion of F4/80+CD86+bone marrow-derived macrophages was significantly lower than that in the high-glucose control group(P<0.05).(2)qRT-PCR results showed that with the treatment of 160,320,and 640 μmol/L mogroside V,the mRNA expression levels of inducible nitric oxide synthase and interleukin 6 were significantly lower than that in the high-glucose control group(P<0.05).With the treatment of 320 and 640 μmol/L mogroside V,the mRNA expression level of interleukin 1β was significantly lower than that in the high-glucose control group(P<0.05).(3)ELISA results exhibited that with the treatment of 160,320,and 640 μmol/L mogroside V,the tumor necrosis factor-α secretion level was significantly lower than that in the high-glucose control group(P<0.05).(4)With the treatment of 320 μmol/L mogroside V,calcium salt deposition was increased in bone marrow mesenchymal stem cells under high glucose and inflammatory conditions(P<0.05),and the mRNA relative expression levels of alkaline phosphatase,Runt-related factor 2,and osteopontin were increased(P<0.05).These findings indicate that mogroside V can promote osteogenic differentiation of bone marrow mesenchymal stem cells by inhibiting the M1 polarization of bone marrow-derived macrophages under high glucose and inflammatory conditions and reducing the generation of inflammatory factors.

[摘 要] 生长分化因子15（GDF15）是介导多种肿瘤耐药的关键因子。它主要通过激活PI3K/AKT-Nrf2等信号通路调控肿瘤细胞凋亡逃逸与代谢重编程并参与诱导EMT和重塑免疫微环境等机制，使肿瘤细胞的耐药能力增强。尽管GDF15相关信号通路的多样性及受体依赖性的差异给精准靶向治疗带来了挑战，但GDF15在肿瘤患者血清中的表达显著升高表明了它作为预后标志物和治疗靶点的较高临床转化潜力。本文整合GDF15与不同信号通路的关联及其在肿瘤耐药中的作用机制后认为，将联合靶向策略、PROTAC技术的应用以及动态监测体系的建立作为未来研究方向，有望为治疗GDF15介导的肿瘤化疗耐药提供有效方案，进而提升相关抗耐药治疗的临床应用价值。

Objective:To construct the sphingosine kinase 1(SPHK1)overexpression lentiviral vector,and to establish the SKOV3 lentiviral stable transfection cell line.Methods:According to the SPHK1 data information provided by the National Center for Biotechnology Information(NCBI)database,the primers were designed and synthesized,the target gene was amplified,and connected to the GV492 plasmid treated with Bam HⅠ and AgeⅠ restriction enzymes to construct the SPHK1 overexpression lentiviral vector;the positive clones were selected for PCR and sequencing identification;the lentiviral plasmid and the lentiviral packaging auxiliary plasmid were co-transfected into the HEK-293T cells for packaging and titer determination;according to the measured optimal multiplicity of infection(MOI)of 10,the corresponding lentiviral amounts in various groups were transfected into the SKOV3 cells,and the SKOV3 cells were divided into blank group(without treatment),GV492 control group(GV492 control lentivirus infected SKOV3 cells),and GV492-SPHK1 overexpression group(GV492-SPHK1 overexpression lentivirus infected SKOV3 cells,ov-SPHK1 group);the optimal concentration of 2 mg·L-1 puromycin was used to screen the stably transfected SKOV3 cell line;after 48 h,the medium was changed and replaced with 1 mg·L-1 puromycin for screening for 14 d;the morphology and fluorescence expression of the cells were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of SPHK1 mRNA in the SKOV3 cells in various groups;Western blotting method was used to detect the expression level of SPHK1 protein in the SKOV3 cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the SPHK1 overexpression lentiviral vector was completely consistent with the target sequence,and the SPHK1 overexpression lentiviral vector was successfully constructed;the titer determination results showed that the lentiviral titers in GV492 control group and ov-SPHK1 group were 5×1011 and 8×1011 TU·L?1,respectively;the SKOV3 cells in GV492 control group and ov-SPHK1 group were in good state and showed strong fluorescence expression,suggesting that the SKOV3 stable transfection cell line overexpressing SPHK1 was successfully established;the RT-qPCR results showed that compared with blank group and GV492 control group,the expression level of SPHK1 mRNA in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01);the Western blotting results showed that compared with blank group and GV492 control group,the expression level of SPHK1 protein in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01).Conclusion:The SPHK1 overexpression lentiviral vector is successfully constructed,and the SKOV3 stable transfection cell line is established.

Objective To analyze risk factors for sleep disorders in patients with chronic heart failure(CHF)and construct a nomogram prediction model.Methods Using simple random sampling,306 hospital-ized CHF patients meeting inclusion criteria were enrolled from four Grade A tertiary hospitals in Guangxi Zhuang Autonomous Region(two in Nanning,one each in Yulin and Guilin)between March 2023 and March 2024.LASSO regression analysis was initially employed for variable screening,followed by logistic regression to identify predictive variables for constructing the nomogram model.Model validation and performance evalua-tion were conducted using receiver operating characteristic(ROC)curves,calibration curves,and clinical decision curves,with internal validation performed through Bootstrap resampling(1 000 iterations).Results The incidence of sleep disorders among the 306 patients was 57.5%(176/306).Logistic regression analysis identified eight independent risk factors for sleep disorders in CHF patients(P＜0.05):age,education level,monthly house-hold income per capita,NYHA cardiac function classification,number of comorbidities,triglyceride levels,ano-rexia,and anxiety.The model demonstrated good discrimination for the AUC of 0.91(95%CI:0.77-0.88)and calibration consistency.Conclusion The prediction model established in this study shows good predictive performance,serving as a valuable reference for healthcare providers to early identify sleep disorders and im-plement preventive care strategies in patients with CHF.

Objective To study the effects of cognitive behavioral therapy(CBT)on nutritional status and postoperative recovery in patients after gastric cancer surgery.Methods Sixty patients diagnosed with gastric cancer between January 1,2023,and December 30,2023,at the hospital in the department of gastroin-testinal and gland surgery were included in this study.Patients were randomly divided by drawing lots into ei-ther the observation group(received routine care plus CBT—based nursing program)or the control group(received routine care),with 30 patients in each group.Nutritional indicators and postoperative recovery out-comes were compared between the two groups.Results After the intervention,patients in the observation group showed significantly higher levels of albumin and hemoglobin compared to the control group(P＜0.05).However,there was no statistically significant difference in prealbumin levels between the two groups(P＞0.05).Furthermore,patients in the observation group scored higher on the Quality of Recovery-40 scale in terms of emotional state,physical comfort,psychological support,and overall score compared to the control group(P＜0.05).After the intervention,the observation group showed significantly lower HADS scores com-pared to the control group,with a statistically significant difference(P＜0.05).Conclusion CBT demon-strates significant positive effects in improving nutritional status and postoperative recovery quality in gastric cancer patients.CBT improves patients'emotional state,thereby affecting appetite and nutritional status,and promotes postoperative physical function recovery.

Objective To investigate the effect of body position angle on gastric insufflation during the induction of general anesthesia in pediatric patients undergoing day surgery.Methods A total of 111 children scheduled for elective tracheal intubation under general anesthesia at the First Affiliated Hospital of Guangxi Medical University from December 2022 to March 2023 were selected as study subjects.Six children were ex-cluded due to unclear sonographic visualization of the gastric antrum,resulting in 105 children ultimately in-cluded in the study.The children were divided into three groups using a random number table method:Group D0(supine position),Group D5(5° head-up position),and Group D10(10° head-up position),with 35 children in each group.The presence of a"comet-tail artifact"on ultrasound was used as the criterion for determining gastric insufflation during anesthesia induction.The gastric antrum cross-sectional area(CSA)of the gastric antrum was measured before mask ventilation after loss of consciousness(T1)and immediately after tracheal intubation following mask ventilation(T2).The incidence of gastric insufflation,changes in CSA and their differences,and vital sign changes at T1 and T2,were compared among the three groups.Results Compared with Group D0,the incidence of gastric insufflation was significantly lower in Groups D5 and D10[25.7％(9/35)vs.20.0％(7/35)vs.54.0％(19/35)],and the difference was statistically significant(P＜0.05).Howev-er,there was no significant difference between Group D5 and Group D10(P＞0.05).No significant difference was observed in CSA at T1 among the three groups(P＞0.05).At T2,a statistically significant difference in CSA was found among the three groups(P＜0.05),with Group D10 showing a smaller CSA than Group D0(P＜0.05).The difference in CSA changes was statistically significant among the three groups(P＜0.05),with Groups D5 and D10 exhibiting smaller changes than Group D0,and Group D10 showing a smaller change than Group D5(P＜0.05).No significant differences were observed in mean artery pressure(MAP),heart rate,pulse oxygen saturation(SpO2)and pressure of end-tidal carbon dioxide(PETCO2)at T1 and T2 among the three groups(P＞0.05).A total of 6 children(5.7％)required brief adjustments in jaw support due to irregular PETCO2 waveforms or abnormal peak airway pressure.Conclusion In pediatric patients undergoing day sur-gery under general anesthesia,a 5° head-up position during induction significantly reduces the incidence of gas-tric insufflation and minimizes changes in gastric antral CSA,while a 10° head-up position does not provide ad-ditional benefits.

Objective To explore the perioperative safety and prognostic factors of sequential hepatectomy after conversion therapy using vascular interventional therapy(including transarterial chemoembolization and hepatic arterial infusion chemotherapy)combined with tyrosine kinase inhibitors(TKI)and programmed death-1(PD-1)inhibitors in patients with initially unresectable hepatocellular carcinoma.Methods The clinical data of 106 eligible HCC patients treated in Tumor Hospital Affiliated to Guangxi Medical University from Nov.2019 to Apr.2024 were retrospectively analyzed.The perioperative parameters and postoperative pathological outcomes were described in detail,and factors influencing prognosis were analyzed.Results The median operative time for hepatectomy after conversion therapy was 240 min,with a median blood loss of 200 mL.Intraoperative blood transfusion was required in 24(22.6％)patients.Postoperative adverse reactions occurred in 49.1％(52/106)of patients,with liver failure being the most common adverse reactions(23 patients,21.7％).One(0.9％)patient died during the perioperative period,while the remaining 105 patients were followed up for a median duration of 14.7 months,during which 49(46.2％)patients experienced recurrence.Among them,39(36.8％)cases experienced early recurrence(within 1 year),and 33(31.1％)cases had intrahepatic recurrence.Thirteen(12.3％)patients died during follow-up.The median recurrence-free survival(RFS)was 15.7 months,with 1-year and 2-year RFS rates being 56.9％and 40.3％,respectively.The median overall survival(OS)was not reached,with 1-year and 2-year OS rates being 94.2％and 85.3％,respectively.Multivariate Cox regression analysis demonstrated that achieving complete pathological response(hazard ratio[HR]=0.410,95％confidence interval[CI]0.172-0.980,P=0.045),presence of microvascular invasion(HR=2.423,95％CI 1.269-4.625,P=0.007),satellite nodules(HR=1.916,95％CI 1.014-3.620,P=0.045),and multiple tumors(HR=1.818,95％CI 1.012-3.241,P=0.046)were independent factors associated with postoperative recurrence.Conclusion For patients with initially unresectable hepatocellular carcinoma,vascular interventional therapy combined with TKI and PD-1 inhibitors followed by sequential hepatectomy may be a feasible treatment strategy,with manageable adverse reactions and promising efficacy.