BACKGROUND:Bone relies on the close connection between blood vessels and bone cells to maintain its integrity.Bones are in a physiologically hypoxic environment.Therefore,the study of angiogenesis and osteogenesis in hypoxic environment is closer to the microenvironment in vivo. OBJECTIVE:To explore the influence of Wenshen Tongluo Zhitong(WSTLZT)formula on H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell co-culture system in hypoxia environment and its related mechanism. METHODS:Enzyme digestion method and flow sorting technique were used to isolate and identify H-type bone microvascular endothelial cells.Mouse bone marrow mesenchymal stem cells were isolated and obtained by bone marrow adhesion method.H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell hypoxic co-culture system was established using Transwell chamber and anoxic culture workstation.WSTLZT formula powder was used to intervene in each group at a mass concentration of 50 and 100 μg/mL.The angiogenic function of H-type bone microvascular endothelial cells in the co-culture system was evaluated by scratch migration test and tube formation test.The osteogenic differentiation ability of bone marrow mesenchymal stem cells in the co-cultured system was evaluated by alkaline phosphatase staining and alizarin red staining.The protein and mRNA expression changes of PDGF/PI3K/AKT signal axis related molecules in H-type bone microvascular endothelial cells in the co-cultured system were detected by Western Blotting and q-PCR,respectively. RESULTS AND CONCLUSION:(1)Compared with the normal oxygen group,the scratch mobility and new blood vessel length of H-type bone microvascular endothelial cells were significantly higher(P<0.05);the osteogenic differentiation capacity of bone marrow mesenchymal stem cells was higher(P<0.05);the expression of PDGF/PI3K/AKT axis-related molecular protein and mRNA increased(P<0.05)in the hypoxia group.(2)Compared with the hypoxia group,scratch mobility and new blood vessel length were significantly increased in the H-type bone microvascular endothelial cells(P<0.05);bone marrow mesenchymal stem cells had stronger osteogenic function(P<0.05);the expression of PDGF/PI3K/AKT axis-related molecular proteins and mRNA further increased(P<0.05)after treatment with different dose concentrations of WSTLZT formula.These findings conclude that H-type angiogenesis and osteogenesis under hypoxia may be related to the PDGF/PI3K/AKT signaling axis,and WSTLZT formula may promote H-type vasculo-dependent bone formation by activating the PDGF/PI3K/AKT signaling axis,thereby preventing and treating osteoporosis.

The doctor-patient relationship is a set of social relationships based on shared interests， mutual trust， and emotional bonds， to relieve illnesses and promote health. However， the doctor-patient relationship often falls into tensions and conflicts. How to build a trusting and harmonious doctor-patient destiny community has become one of the most important issues of concern to the whole society. Based on the biopsychosocial concept of disease， the emotion management technique （EMT） emphasizes that doctors take the patient’s emotion as a clue in clinical diagnosis and treatment， regard emotions as one of the important indicators for disease diagnosis， understand the emotional events behind the disease， and provide patients with appropriate empathy and emotional management， so as to provide clinical methods for managing diseases and building a trusting and harmonious doctor-patient relationship.

Objective To analyze the expression levels of RNA demethylase alkB homolog 5(ALKBH5)and p21 activated kinase 5(PAK5)in high-risk human papillomavirus(HR-HPV)positive cervical cancer tissue,and explore their clinical significance.Methods A total of 96 patients with HR-HPV positive cervical cancer were selected as the research object.The expressions of ALKBH5 and PAK5 mRNA and protein in cancer tissue and adjacent tissue were analyzed by real-time fluorescent quantitative PCR,immunohistochemistry and Western blot assay.The expression levels of ALKBH5 and PAK5 proteins in patients with HR-HPV positive cervical cancer with different clinicopathological characteristics were compared.Multivariate Cox regression analysis was used to analyze influencing factors of death in patients with HR-HPV positive cervical cancer.Kaplan-Meier curve was used to analyze the effects of ALKBH5 and PAK5 protein expression on the 5-year survival rate of patients.Results The results of real-time fluorescent quantitative PCR,immunohistochemistry and Western blot assay showed that expression levels of ALKBH5 and PAK5 mRNA and protein in HR-HPV positive cervical cancer tissue were higher than those in adjacent tissue(P<0.05).The positive rates of ALKBH5 and PAK5 in cancer tissue of HR-HPV positive cervical cancer patients with FIGO stage ⅡA and lymph node metastasis were increased(P<0.05).ALKBH5 positive(HR=1.467,95％CI:1.104-1.949),PAK5 positive(HR=1.435,95％CI:1.093-1.884),FIGO stage ⅡA(HR=1.435,95％CI=1.065-1.933)and lymph node metastasis(HR=1.652,95％CI:1.053-2.593)were risk factors affecting the prognosis of HR-HPV positive cervical cancer patients(P<0.05).The 5-year cumulative survival rate was lower in the ALKBH5 positive group[72.58％(45/62)vs.91.18％(31/34)]than that of the ALKBH5 negative group(log rank χ2=4.481,P=0.034).The 5-year overall survival rate of the PAK5 positive group[71.67％(43/60)vs.91.67％(33/36)]was lower than that of the PAK5 negative group(log rank χ2=4.153,P=0.043).Conclusion The expression levels of ALKBH5 and PAK5 are significantly up-regulated in HR-HPV positive cervical cancer,both of which are related to the poor clinicopathological features of patients,and are helpful to evaluate the prognosis.

Objective:To evaluate the role of fibrinogen in perioperative neurocognitive disorder (PND) in aged mice.Methods:Sixty SPF healthy male C57BL/6J mice, aged 16-18 months, weighing 25-30 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), PND group (group P), urokinase group (group U) and PND+ urokinase group (group PU). Abdominal surgery was performed under 3% sevoflurane anesthesia to establish the mouse model of PND. In PU group, urokinase 20 000 U/kg was intraperitoneally administered at 1 h after surgery, once a day, for 5 consecutive days. In group U, urokinase was intraperitoneally injected once a day for 5 consecutive days without anesthesia and surgery. The cognitive function was assessed after operation using the novel object recognition test (discrimination index) and the Morris water maze test (frequency of crossing the original platform and percentage of the time spent in the target quadrant). The expression of occludin, claudin-5, fibrinogen and ionized calcium-binding adapter molecule 1 (Iba-1) and CD11b in hippocampal tissues was detected using Western blot, the area of fibrinogen extravascular deposits was measured and the morphology of microglia was observed using the immunofluorescence staining, and the mRNA expression of pro-inflammatory factors (interleukin-1beta, tumor necrosis factor-alpha, and inducible nitric oxide synthase), anti-inflammatory factors (interleukin-4 and arginase-1), and chemokines (chemokine 2 and chemokine ligand 10) in hippocampal tissues was detected by quantitative real-time polymerase chain reaction after surgery. Results:Compared with group C, the parameters of cognitive function were significantly decreased, the expression of occludin and claudin-5 was down-regulated, the expression of fibrinogen was up-regulated, the area of fibrinogen extravascular deposits was increased, the number of branches was decreased and the average process length was shortened in the microglia around fibrinogen deposits, the expression of Iba-1 and CD11b was up-regulated, the mRNA expression of proinflammatory cytokines and chemokines was up-regulated, and the mRNA expression of the anti-inflammatory factors was down-regulated in group PND ( P<0.05). Compared with group PND, the parameters of cognitive function were significantly increased, the expression of occludin and claudin-5 was up-regulated, the expression of fibrinogen was down-regulated, the area of fibrinogen extravascular deposits was decreased, the number of branches was increased and the average process length was prolonged in the microglia around fibrinogen deposits, the expression of Iba-1 and CD11b was down-regulated, the mRNA expression of proinflammatory cytokines and chemokines was down-regulated, and the mRNA expression of the anti-inflammatory factors was up-regulated in group PU ( P<0.05). Conclusions:Fibrinogen deposits in the brain parenchyma through the damaged blood-brain barrier after anesthesia and surgery and participates in the development of PND, and the underlying mechanism may be related to the promotion of microglial activation and the induction of neuroinflammation in aged mice.

Objective:To evaluate the relationship between the mechanism of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) preventing sevoflurane-induced neurotoxicity and phosphorylated Tau glymphatic system clearance pathway in neonatal mice.Methods:Eighteen C57BL/6 pregnant mice were used in this study and subjected to 2 feeding regiments using the random number table method. Twelve mice were selected to receive a standard diet, and 6 mice were selected to receive a diet supplemented with fish oil (ω-3 polyunsaturated fatty acids [300 mg was added to every 20 g of standard diet from the 2nd day of gestation to 14 days after parturition). The healthy neonatal mice of both sexes, aged 6 days, weighing 3-5 g, were selected after parturition. Forty-eight neonatal pups from 6 pregnant mice that were fed a standard diet were assigned to control group (C group), 48 neonatal pups from 6 pregnant mice that were fed a standard diet were assigned to sevoflurane group (S group), and 48 neonatal pups from pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus sevoflurane group (PS group) using the random number table method. All the offspring mice in all groups were breastfed until 21 days of birth and then were housed in separate cages from their mothers after 21 days of birth and provided with ad libitum access to standard food. S group and PS group inhaled 3% sevoflurane and 40% oxygen for 2 h daily on postnatal days 6, 7 and 8. C group inhaled only 40% oxygen at the same flow rate. Y maze test was performed at postnatal day 33 to assess the spatial memory and cognitive function. The rotarod test was performed at postnatal day 35 to assess the fine motor coordination. The influx and efflux functions of the glymphatic system were assessed through intracisternal tracer infusion with the fluorescent tracer at postnatal days 14 and 35. The influx function was evaluated by the percentage of the area of tracer penetration 30 min after injection, while the efflux function was determined by the percentage of the residual area of the tracer 90 min after injection. The mice were sacrificed and the hippocampal tissue was obtained at postnatal day 14 for determination of the expression of phosphorylated Tau protein at serine 202 site and threonine 205 site (Tau-PS202/PT205) and total Tau protein by Western blot. The cerebrospinal fluid (CSF) was collected at postnatal day 14 for determination of the concentration of phosphorylated Tau protein by enzyme-linked immunosorbent assay. The mice were sacrificed and the hippocampal tissue was obtained at postnatal day 35 for determination of the expression of caspase-3, caspase-9 and cytochrome C (Cyt c) (by Western blot) and the apoptosis rate of neurons (by TUNEL).Results:Compared with C group, the time of staying at the new arm and in the rotarod test was significantly shortened, the percentage of new arm movement distance was decreased, the percentage of tracer penetration area was decreased at postnatal day 14, the percentage of residual tracer area was increased at postnatal day 14, the expression of Tau-PS202/PT205 in the hippocampus was up-regulated at postnatal day 14, the concentration of phosphorylated Tau protein in CSF was reduced at postnatal day 14, the apoptosis rate of hippocampal neurons was increased at postnatal day 35 ( P<0.05), and the expression of caspase-3, caspase-9 and Cyt c in the hippocampus was up-regulated at postnatal day 35 in S group ( P<0.05). Compared with S group, the time of staying at the new arm and in the rotarod test was significantly prolonged, the percentage of new arm movement distance was increased, the percentage of tracer penetration area was increased at postnatal day 14, the percentage of residual tracer area was decreased at postnatal day 14, the expression of Tau-PS202/PT205 in the hippocampus was down-regulated at postnatal day 14, the concentration of phosphorylated Tau protein in CSF was increased at postnatal day 14, the apoptosis rate of hippocampal neurons was decreased at postnatal day 35, and the expression of caspase-3, caspase-9 and Cyt c in the hippocampus was down-regulated at postnatal day 35 in PS group ( P<0.05). Conclusions:The mechanism by which ω-3 PUFAs prevents cerebral neurotoxicity induced by repeated neonatal sevofurane exposure may be related to the enhancement of phosphorylated Tau protein clearance via the glymphatic system.

Objective:To evaluate the relationship between the mechanism of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) in preventing brain neurotoxicity caused by multiple sevoflurane anesthesia and peroxisome proliferator-activated receptor gamma (PPARγ)/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) signaling pathway in neonatal mice.Methods:This study was performed in 2 parts. Part Ⅰ Using a random number table method, 6 C57BL/6 pregnant mice were assigned to receive a standard diet, 3 pregnant mice were assigned to receive a diet supplemented with fish oil from day 2 of gestation to day 14 after parturition (ω-3 PUFAs 300 mg were added to every 20 g of conventional diet). Healthy C57BL/6 mice of both sexes, aged 6 days, weighing 3-5 g, were selected after parturition. Seventeen neonatal pups from 3 pregnant mice that were fed a conventional diet were assigned to control group (C group), 17 neonatal pups from 3 pregnant mice that were fed a conventional diet were assigned to sevoflurane group (S group), and 17 neonatal pups from pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus sevoflurane group (PS1 group) using the random number table method. Part Ⅱ Four C57BL/6 pregnant mice were assigned to receive a diet supplemented with fish oil from day 2 of gestation to day 14 after parturition. After parturition, 12 neonatal pups from 2 pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus sevoflurane group (PS2 group), and 12 neonatal pups from 2 pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus PPARγ inhibitor GW9662 plus sevoflurane group (PGS group) using a random number table method. GW9662 (2 mg/kg) was intraperitoneally injected at 30 min before exposure to sevoflurane in PGS group. All offspring mice were breastfed until 21 days of age, after which they were housed separately from the mother and allowed ad libitum access to a conventional diet. S, PS1, PS2 and PGS groups inhaled 3% sevoflurane in 40% oxygen for 2 h daily on postnatal days 6, 7 and 8. C group inhaled only 40% oxygen instead. Y maze test was performed on days 33 after birth. The rotarod test was performed on day 35 after birth. After the behavioral testing, the expression of PPARγ, PGC1α, mitofusin-1 (MFN1), MFN2, dynamin-related protein 1 (DRP1), interleukin-6 (IL-6), interleukin-1 β (IL-1 β), and tumor necrosis factor-α (TNF-α) was detected by Western blot, the ultrastructure of mitochondria in hippocampal neurons was observed with a transmission electron microscope, and the mitochondrial membrane potential (MMP), level of reactive oxygen species (ROS) and content of ATP were determined.Results:Part Ⅰ Compared with C group, the time of stay at the new-arm and time spent on the rotarod were significantly shortened, the percentage of movement distance in the new-arm was decreased, the expression of PPARγ, PGC1α, MFN1 and MFN2 in the hippocampus was down-regulated, the expression of DRP1, IL-6, IL-1β and TNF-α in the hippocampus was up-regulated, the MMP and content of ATP were decreased, and the level of ROS was increased in S group ( P<0.05). Compared with S group, the time of stay at the new-arm and time spent on the rotarod were significantly prolonged, the percentage of movement distance in the new-arm was increased, the expression of PPARγ, PGC1α, MFN1 and MFN2 in the hippocampus was up-regulated, the expression of DRP1, IL-6, IL-1β and TNF-α in the hippocampus was down-regulated, the MMP and content of ATP were increased, and the level of ROS was decreased in PS1 group ( P<0.05). Part Ⅱ Compared with PS2 group, the time of stay at the new-arm and time spent on the rotarod were significantly shortened, the percentage of movement distance in the new-arm was decreased, the MMP and content of ATP were decreased, the level of ROS was increased, and the expression of IL-6, IL-1β and TNF-α was up-regulated ( P<0.05). Conclusions:The mechanism by which ω-3 PUFAs prevent brain neurotoxicity caused by multiple sevoflurane anesthesia is related to the activation of the PPARγ/PGC1α signaling pathway and alleviation of mitochondrial dysfunction and neuroinflammation in neonatal mice.

Spleen-stomach damp-heat syndrome is currently the most prevalent TCM pattern in patients with chronic atrophic gastritis (CAG), with internal damp-heat accumulation regarded as a key factor contributing to its prolonged and refractory course. This syndrome represents a critical stage in the progressive pathogenesis of CAG, characterized by a deepening pathological evolution. Modern TCM practitioners generally agree that its core pathogenesis lies in "deficiency in root and excess in superficiality, with internal damp-heat retention", and emphasize a treatment strategy that combines eliminating pathogenic factors and reinforcing the body's healthy qi through dynamic syndrome differentiation. Chinese materia medica used in treating CAG with spleen-stomach damp-heat syndrome can effectively relieve clinical symptoms, improve the internal damp-heat environment, mitigate gastric mucosal atrophy and intestinal metaplasia, and delay the inflammation-to-cancer transformation. Its mechanisms may involve eradication of Helicobacter pylori, repair of gastric mucosal injury, regulation of immune inflammatory response and other aspects, which has the advantages of multi-channel and multi-target.

Objective To identify independent predictors of prognosis in MMNS and to examine the expression of CD4,CD8,CD20 and their relationship with prognosis.Methods 87 patients with MMNS were retrospectively an-alyzed to summarize their clinicopathologic characteristics;the expression of CD4,CD8,and CD20 in MMNS were observed by EnVision immunohistochemical staining(IHC)to analyze the relationship between the expression of these markers and prognosis.Results MMNS with sinus,surrounding tissue invasion,larger tumor and higher AJCC stage showed a worse prognosis.No significant correlation was found among sex,age,histological type,melanin granules,mitotic figures and survival rate.The counts of CD4+,CD8+and CD20+TILs in intra-tumoral stroma were positively correlated with the density of TILs at the base of the tumor.CD20+B cells did not have a central tendency to aggregate to form tertiary lymphoid structures,whether in the intratumoral stroma or in the basal stroma of the tumor,the three cell subsets were admixed.There was no statistically significant difference in OS be-tween CD4+,CD8+,and CD20+subsets.Conclusions The number of CD20+B cells was significantly less than that of CD4+and CD8+T cells in both the intratumoral stroma and the basal stroma of the tumor,and tertiary lymphoid structures fail to be formed,which,unlike cutaneous malignant melanoma,might be a cause of the poor prognosis of MMNS.

Objective To investigate the expression levels of serum hemoglobin β(HBB)in hepatocellular carcinoma(HCC)patients and its correlation with topoisomeraseⅡα(TOP2A)gene.Methods A total of 48 HCC patients visited the Second Affiliated Hospital of Nantong University from August 2023 to September 2024 were selected as the HCC group,and 32 healthy individuals who underwent physical examination during the same period were selected as the healthy control group.Their blood samples were collected,and the ex-pression levels of serum HBB and TOP2A genes were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Then,the relationship between the expression of HBB gene and clinicopathological parameters of the patients was ana-lyzed.The Kaplan-Meier Plotter database was used to evaluate the relationship between the expression of HBB gene and the prognosis of HCC.The diagnostic value of the expressions of serum HBB and TOP2A genes for HCC was assessed by the receiver operating charac-teristic(ROC)curve.Spearman rank correlation analysis was used to evaluate the correlation between the expressions of serum HBB and TOP2A genes in HCC patients.The regulatory effect of HBB gene on TOP2A gene was verified by the cell experiment.Results The expression levels of serum HBB gene in HCC patients(0.097[0.055,0.155])were significantly lower than that in healthy controls(1.029[0.625,1.434],U=19,P＜0.001).The expression levels of serum TOP2A gene in HCC patients(1.810[0.825,3.623])were significantly higher than that in healthy controls(1.047[0.604,1.364],U=495,P=0.007).The expression level of serum HBB gene in HCC patients was significantly negatively correlated with that of TOP2A gene(ρ=-0.384,P=0.007).The analysis results of clinicopathological parameters showed that the expression level of HBB gene was only related to tumor size(χ2=4.090,P＜0.05).The Kaplan-Meier survival analysis showed that the 5-year overall survival rate of the patients with low expression of HBB gene was signifi-cantly lower than that with high expression(HR=0.680,95%CI:0.470-0.970,P＜0.05).The analysis of the ROC curve showed that the area under the ROC curve(AUCROC)of HBB gene in diagnosing HCC was 0.987(95%CI:0.963-1.000).When the cut-off value was 0.228,its sensitivity was 100%and specificity was 97%.The AUCROC of TOP2A gene for the diagnosis of HCC was 0.677(95%CI:0.559-0.797).When the cut-off value was 1.285,its sensitivity was 65%and specificity was 75%.The combined detection of HBB and TOP2A genes for the diagnosis of HCC had an AUCROC of 0.988(95%CI:0.965-1.000).When the cut-off value was 0.657,its sensitivity was 100%and specificity was 97%.The cell experiment results showed that the overexpression of HBB gene could inhibit the expression of TOP2A gene,while the knockout of HBB gene had the opposite effect.Conclusion HBB gene is lowly expressed in the serum of HCC patients and is significantly negatively correlated with the expression of TOP2A gene.

Nonalcoholic steatohepatitis （NASH） is a chronic liver disease with the main pathological features of hepatic steatosis， inflammatory cell infiltration， and interstitial fibroplasia， and it is an important risk factor for liver fibrosis， liver cirrhosis， and hepatocellular carcinoma. NOD-like receptor protein 3 （NLRP3） inflammasome is the core of innate immunity， and the abnormal activation of NLRP3 inflammasome is closely associated with the development and progression of NASH， which involves multiple links such as inflammatory response and oxidative stress. A large number of studies have shown that the active ingredients of traditional Chinese medicine （TCM） and TCM compound prescriptions can improve oxidative stress， regulate lipid metabolism， and alleviate liver inflammation by regulating NLRP3 inflammasome. TCM treatment applied in clinical practice has achieved a good therapeutic effect， while inflammasome is one of the key pathways or targets for TCM in improving NASH. This article reviews the mechanism of action of NLRP3 inflammasome in NASH and the research advances in TCM intervention of NLRP3 inflammasome， in order to provide ideas for the clinical TCM treatment of NASH， as well as reference targets and research directions for the research and development of new TCM drugs.