Recent clinical studies have shown that mutation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in cancer cells may be associated with immunosuppressive tumor microenvironment (TME) and poor response to immune checkpoint blockade (ICB) therapy. Therefore, efficiently restoring PTEN gene expression in cancer cells is critical to improving the responding rate to ICB therapy. Here, we screened an adeno-associated virus (AAV) capsid for efficient PTEN gene delivery into B16F10 tumor cells. We demonstrated that intratumorally injected AAV6-PTEN successfully restored the tumor cell PTEN gene expression and effectively inhibited tumor progression by inducing tumor cell immunogenic cell death (ICD) and increasing immune cell infiltration. Moreover, we developed an anti-PD-1 loaded phospholipid-based phase separation gel (PPSG), which formed an in situ depot and sustainably release anti-PD-1 drugs within 42 days in vivo. In order to effectively inhibit the recurrence of melanoma, we further applied a triple therapy based on AAV6-PTEN, PPSG^@anti-PD-1 and CpG, and showed that this triple therapy strategy enhanced the synergistic antitumor immune effect and also induced robust immune memory, which completely rejected tumor recurrence. We anticipate that this triple therapy could be used as a new tumor combination therapy with stronger immune activation capacity and tumor inhibition efficacy.

BACKGROUND:Recent studies have shown that research on naringin anti-osteoporosis mostly stays in in vitro and in vivo experiments.Understanding the mechanism of related signaling pathways and the expression of related proteins and some specific genes is an important way to deeply understand naringin anti-osteoporosis.At present,traditional Chinese medicine has been confirmed to have a significant role in anti-osteoporosis.Naringin is one of the main active ingredients in Rhizoma Drynariae.Its effectiveness and mechanism of action against osteoporosis have been gradually recognized by scholars,and its clinical and basic research has been gradually emphasized. OBJECTIVE:To analyze and summarize the research progress of naringin in anti-osteoporosis in vitro and in vivo,thereby providing some ideas for the next step to study its related mechanism of action. METHODS:The relevant literatures included in CNKI and PubMed database were searched with the Chinese search terms of"naringin,osteoporosis,traditional Chinese medicine compound,pathogenesis,signaling pathway,bone marrow mesenchymal stem cells,osteoblasts,osteoclasts"in Chinese and English,respectively.The corresponding criteria were established according to the research needs,and finally 69 articles were included for review. RESULTS AND CONCLUSION:Naringin blocks the increase in the number of osteoclasts and adipocytes,the decrease in the number of osteocytes and osteocalcin(+)cells induced by fructose-rich diet,and promotes the secretion of Sema3A from osteoblasts and osteocytes,thereby enhancing local bone formation and inhibiting osteoclast production by activating the Wnt/β-catenin pathway.Naringin is an important way to induce autophagy of osteoblasts,but autophagy-related proteins participate in osteoblast differentiation and bone formation.Lack of autophagy in osteoblasts reduces mineralization and leads to an imbalance in the number of osteoblasts and osteoclasts,which results in bone loss and decreased bone density.The composite scaffold loaded with naringin can be used as a necessary carrier for bone defect repair and has excellent bone repair properties.Naringin can also accelerate the growth of new bone tissue by increasing the local contents of bone morphogenetic protein 2 and vascular endothelial growth factor.Naringin can regulate bone metabolism and inhibit oxidative stress via ERK,PI3K/Akt and Wnt signaling pathways to improve osteoporosis,which can play a good role in preventing and controlling the disease.However,the depth and breadth of the relevant research is insufficient.Based on the mechanism of the current study,we should investigate the specific mechanisms by which naringin regulates different pathways and inter-pathway interactions in the future,which will be beneficial to the multifaceted development of naringin used in the treatment of osteoporosis..

Objective:To prepare rabbit polyclonal antibodies against respiratory syncytial virus (RSV) N protein and use them as the detection antibodies to establish a N-ELISA-based method for rapid detection of neutralizing antibodies.Methods:A plasmid of pET30a-N for the expression of RSV N protein was constructed. After purification, the protein was immunized into New Zealand rabbits to prepare polyclonal antibodies, which were used as the detection antibodies. Positive serum samples were diluted and used to neutralize RSV (100 TCID 50/well). Hep-2 cells were inoculated and cultured, and then the cells were fixed with 80% acetone. ELISA was performed to detect RSV N protein in infected cells. When the absorbance value of a well was below the cut-off value, it was regarded as the positive well in the neutralization test. The highest dilution of a positive well serum was the neutralizing antibody titer. After optimizting the antibody dilution, detection time, cell density and the duration of neutralization, the method for neutralizing antibody detection was established based on N-ELISA. The established method was verified by analyzing the influences of different cell generations and edge effects, and calculating the accuracy, repeatability and precision. The correlation between the established method and microneutralization method was analyzed by detecting human RSV IgG-positive serum. Results:The plasmid pET30a-N was successfully constructed, and the expressed N protein showed high purity and good specificity. After the third immunization, the antibody titer in rabbit serum was 1∶51 200, and the antibodies could specifically bind to RSV. The prepared rabbit anti-RSV N polyclonal antibodies had a titer of 1∶51 200, and showed good specificity. The neutralizing antibodies could be detected on day 4 with the established method, and the duration of neutralization was shortened to 30 min. Cell generations and the position of wells in the 96-well plate (edge well and non-edge well) had no significant effect on the method, and the repeatability, precision and accuracy of the method were good. In the detection of 64 RSV IgG-positive human serum samples by the established method and microneutralization method, the correlation coefficient was 0.929 6, indicating a good positive correlation between the two methods.Conclusions:A N-ELISA-based method for rapid neutralizing antibody detection is successfully established, which can be used to evaluate the serum antibody level after RSV vaccination.

Objective To analyze the relationship between self-factors and seasonal factors in adult patients with recurrent benign paroxysmal positional vertigo(BPPV),and explore the related factors of recurrent BPPV.Methods A total of 815 adult patients with re-current BPPV admitted to Beijing Puren Hospital from February 2015 to January 2022 were included in a retrospective cohort study.The age,gender,and seasonality of onset were analyzed,and the effects of vascular risk factors,calcium and vitamin D supplementation were explored.Results Patients with recurrent BPPV ranged in age from 22 to 88 years,with a median age of 61(55,67)years,and the gender ratio of males to females was 1∶3.01.There were no significant differences in age,age composition and prevalence of vascular risk factors between males and females(P＞0.05).The proportion of calcium and vitamin D supplementation in female patients was higher than that in male patients(P＜0.001).The occurrence of recurrent BPPV had no significant seasonal regularity,but showed an increas-ing trend in cold season.Vascular risk factors were correlated with age,but not with gender or season.The patients with multiple recur-rence of BPPV accounted for 20.86％(170/815),calcium and vitamin D supplementation were independent influencing factors.Conclusion Recurrent BPPV occurs frequently in females,and is more common in old age,and is more frequent in cold season.Calci-um and vitamin D supplementation may reduce the recurrence frequency.

Objective:To explore the neuroprotective effects and mechanisms of memantine on sepsis-associated encephalopathy (SAE) model mice.Methods:Totally 90 male C57BL/6J mice aged 8-12 weeks were randomly divided into 3 groups (with 30 mice in each group) : sham group, model group and memantine group. The SAE mouse model was established by cecum ligation and puncture while mouse in sham group received open and closed abdomen only. The mice in the memantine group were irrigation with memantine (15 mg · kg -1· d -1) 3 hours before surgery and 7 consecutive days after modeling. The mice in the model group and sham group were irrigation with an equal volume of 0.9% sodium chloride solution. The 7-day survival rate was observed, neurobehavioral and cognitive function scores of each group of mice after modeling were assessed.Blood-brain barrier permeability was measured by detecting the content of Evans blue. Immunofluorescence staining was used to detect the expression of astrocytes. Enzyme linked immunosorbent assay was conducted to detect cellular inflammatory factors and the glutamic acid content detection kit was used to detect the expression of glutamic acid. All data were analyzed by Graphpad Prism 8.3.0 software, survival rate was analyzed using Kaplan-Meier survival curve.Multigroup comparisons were conducted by one-way ANOVA or Kruskal-Wallis test. Results:(1) There was a statistically significant difference in the 7-day survival rate among the three groups of mice after modeling ( F=24.11, P<0.01), and the 7-day survival rate of the memantine group was higher than that of the model group (57% (17/30), 27% (8/30), P<0.01). (2)The behavioral results showed that after 7 days of modeling, there were statistically significant differences in the total distance of the open field test, central area stay time, four corner area stay time, neurobehavioral scores, pole climbing test, and preference index for new object recognition test among the three groups of mice ( F/ χ2=17.67, 17.30, 9.39, 14.06, 10.36, 14.81, all P<0.05).The neurobehavioral score, pole climbing test score, preference index for new object recognition test, total distance of open field test, and central area stay time of the model group were all lower than those of the sham group (all P<0.05), while four corner area stay time of the model group was higher than that of the sham group ( P<0.05).The total distance of open field test (1 564.07(1 363.24, 1 988.19) cm, 913.91 (574.32, 1 096.23) cm), central area stay time (5.21 (4.91, 8.76) s, 1.09 (0.25, 1.64) s), neurobehavioral scores (9.75±0.50, 8.25±0.50), pole climbing test scores (5.67±0.52, 4.56±0.53), and preference index for new object recognition test (56.50±10.59, 26.84±2.91) of the memantine group were all higher than those of the model group (all P<0.05). The four corner area stay time was lower than that of the model group ((480.30±50.64) s, (529.80±36.20) s, P<0.05).(3)The comparison of molecular indicators showed that there were statistically significant differences in the content of Evans blue in the brain, the number of astrocytes in the hippocampus and cerebral cortex, pro-inflammatory cytokines (TNF-α、IL-1β、IL-6), anti-inflammatory cytokines (IL-10), and glutamic acid among the three groups of mice ( F/ χ2=8.84, 6.43, 28.46, 23.63, 12.23, 16.04, 69.22, 6.65, all P<0.05).The content of Evans blue, the number of astrocytes in the hippocampus and cerebral cortex, the expression of TNF-α、IL-1β、IL-6, and glutamate in the model group were all lower than those in the sham group(all P<0.05). The levels of IL-10 in the model group was lower than that in the sham group ( P<0.05).The content of Evans blue ((5.67±1.38)μg/g, (11.08±2.79)μg/g), the number of astrocytes in the hippocampus (16.50 (13.75, 22.25)/μm 2), 80.00 (73.50, 83.50)/μm 2) and the cerebral cortex (40.00 (29.00, 48.00)/μm 2, 81.50 (72.25, 89.00)/μm 2) in the memantine group were lower than those in the model group (all P<0.05).The pro-inflammatory factor TNF-α, IL-1β, IL-6 and glutamic acid expression in the memantine group were lower than those in the model group (all P<0.05), and the anti-inflammatory cytokine IL-10 was higher than that in the model group ( P<0.05). Conclusions:Memantine can improve the neurobehaviors and cognitive functions of SAE mice through improving the integrity of the damaged blood-brain barrier, alleviating inflammation in the brain, as well as reducing glutamate levels in the brain.

Background:Celiac disease has a wide range of clinical manifestations and a higher prevalence in women.Aims:To evaluate the differences in clinical characteristics in adult celiac disease patients with different genders.Methods:Adult patients(age≥18 years)diagnosed as celiac disease from July 2017 to July 2022 at the People's Hospital of Xinjiang Uygur Autonomous Region were included consecutively in the retrospective study.Comparisons of baseline demography,and clinical and pathological features between different genders were performed.Results:A total of 73 adult celiac disease patients were enrolled,19 were males and 54 were females,with the ratio of male to female being 1:2.84.The peak age group of onset was 30-59 years old,with an average age of(50.2±13.6)years for men and(43.5±13.2)years for women at diagnosis.There was no significant difference in the distribution of different age groups between men and women(P>0.05),but the proportion of women in 18-49 years age group and≥50 years age group were both higher than that of men with statistical significance(P<0.05).The most common gastrointestinal symptoms were chronic diarrhea(56.2%)and abdominal pain(56.2%),and anemia was the most common extraintestinal manifestation(50.7%),which was existed in 36.8%of males and 55.6%of females.Abdominal pain and nausea/vomiting were more common in females(all P<0.05),whereas no statistically significant differences were found between male and female groups in other gastrointestinal and extraintestinal manifestations,such as chronic diarrhea,flatulence,decreased appetite,weight loss,chronic fatigue,anemia,hypoproteinemia,osteoporosis/bone loss,etc(all P>0.05).The pathological grading of small intestinal biopsy was Marsh Ⅱin 6 cases,and Marsh Ⅲa,Ⅲb and Ⅲc in 14,23,and 30 cases,respectively.No statistically significant difference was observed in pathological grading between patients with different genders(P>0.05).Conclusions:There is a marked female predominance in adult celiac disease in Xinjiang Uygur Autonomous Region,and women were diagnosed at a younger age.Chronic diarrhea,abdominal pain and anemia present as the most common gastrointestinal and extraintestinal manifes-tations.Abdominal pain and nausea/vomiting are more common in women than in men.

Objective:To explore whether barium chloride (BaCl 2) preconditioning has the protective effect on lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) model in mice and the possible mechanism. Methods:Sixty 8-12 week old healthy C57BL/6 male mice were randomly divided into control group, ARDS model group and BaCl 2 pretreatment group, with 20 mice in each group. The BaCl 2 pretreatment group was continuously injected with BaCl 2 (4 mg/kg through the tail vein) for 3 days before ARDS model establishment. ARDS model was established by intratracheally injecting (3 mg/kg) LPS. The control group was intratracheally given the same volume of 0.9% normal saline. On 24th hour after ARDS model establishment, some mice were sacrificed for obtaining fresh lung tissue. And the right lower lobe of the lung was separated for observing the pathological changes of lung tissue while the left lung tissue was used to measure the wet/dry weight ratio (W/D) of the lung. Some mice were sacrificed for observing pulmonary microvascular permeability at 2nd hours after injecting Evans blue (EB) through tail vein. The left mice were killed for alveolar lavage to measure the levels of tumor necrosis factor-α (TNF-α) via enzyme linked immunosorbent assay (ELISA). Results:Comparing with the control group, ARDS model group showed typical ARDS pathological changes, which included the increased W/D ratio (4.951±0.161 vs. 3.449±0.299, P < 0.01) and the content of EB in the lung tissue (μg/g: 0.130±0.027 vs. 0.085±0.011, P < 0.01), the damaged alveolar wall structure, lung congestion and exudates in the alveoli, as well as amounts of inflammatory cells. The pathological score of lung injury (10.33±1.15 vs. 1.67±0.58) and the level of TNF-α in BALF (ng/L: 900.85±247.80 vs. 68.21±5.79) were significantly increased in the ARDS model group (both P < 0.01). Comparing with the ARDS model group, the lung W/D ratio (4.620±0.125 vs. 4.951±0.161) and the EB content in the lung tissue (μg/g: 0.108±0.011 vs. 0.130±0.027) of BaCl 2 pretreatment group were significantly reduced (both P < 0.01). And the damaged pulmonary structural BaCl 2 pretreatment group were significantly alleviated. In addition, the pulmonary pathological score (5.00±1.00 vs. 10.33±1.15) and the level of TNF-α in BALF (ng/L: 169.16±73.33 vs. 900.85±247.80) were significantly decreased (both P < 0.01). Conclusion:Barium chloride pretreatment can improve the lung histopathological changes of ARDS model mice induced by LPS by reducing the permeability of pulmonary capillaries and local inflammatory reaction.Barium chloride has the protective effect against LPS attack in mice model of ARDS.

Objective:To observe the correlation between the frontal P-wave axis and the severity of chronic obstructive pulmonary disease (COPD) and the prognosis evaluation system.Methods:Patients with COPD>45 years old who were followed up in the outpatient department of Hunan Chest Hospital from January to July 2022 were continuously selected as subjects. At the same time, the healthy people who examined in the health management center of our hospital were in the control group. Both groups of subjects completed electrocardiogram and pulmonary function tests. The level of frontal P-wave axis and the results of pulmonary function examination were recorded, and the differences of frontal P-wave axis between the COPD group and the control group were compared, so as to clarify the value of frontal P-wave axis in the diagnosis, disease severity and prognosis evaluation of COPD.Results:The level of forced expiratory volume in the first second/forced vital capacity(FEV1/FVC )in the COPD group was significantly lower than that in the control group, while the level of P-wave axis was significantly higher than that in the control group (all P<0.05). The receiver operating characteristic (ROC) curve of P-wave axis showed that the AUC of P-wave axis in predicting COPD was 0.96 ( P<0.001), the best cut-off value was 63.80, the sensitivity was 0.89, and the specificity was 0.93. There were significant difference in P-wave axis level, the forced expiratory volume in one second to forced vital capacity ratio (FEV 1%pred), body mass index (BMI) and BMI, airflow obstruction, dyspnea, and exercise capacity (BODE) index between groups according to the degree of airflow limitation (all P<0.05). Correlation analysis showed that P-wave axis level was positively correlated with BODE index ( r=0.77, P<0.001), and negatively correlated with pulmonary function FEV 1%pred ( r=-0.76, P<0.001). Conclusions:There is a good correlation between the level of frontal P-wave axis and the severity of COPD and the prognosis evaluation system, which has clinical application value.

To evaluate the association between serum anti-tissue transglutaminase antibody (anti-tTG) titers and the severity of histological damage to the duodenal mucosa and to predict a possible anti-tTG cutoff value for diagnosing celiac disease (CD) and villous atrophy in the domestic population. Clinical and pathological data from 76 adult CD patients with positive anti-tTG titers and duodenal biopsy results who were treated at the People′s Hospital of Xinjiang Uygur Autonomous Region from July 2017 to January 2022 were retrospectively analyzed. The correlation between anti-tTG titers and the severity of duodenal mucosal damage was statistically assessed to predict the optimal anti-tTG titer cut-off value for diagnosing CD and villous atrophy. Of the 76 patients, 10 had underlying CD, and of the 66 patients with duodenal histopathology, four were Marsh Ⅰ, six were Marsh Ⅱ, and 56 were Marsh Ⅲa-c grade. In adults with CD, anti-tTG titers were shown to be associated with the severity of histological damage to the duodenal mucosa. When the anti-tTG level was ≥5 times the upper limit of normal (ULN), the sensitivity and specificity for diagnosing CD were 83.9% and 92.9%, respectively. When the anti-tTG titer was ≥8 times the ULN, the sensitivity and specificity for diagnosing villous atrophy were 67.9% and 90.0%, respectively. Anti-tTG levels had a strong predictive value for diagnosing CD in adults when titers exceeded 10 times the ULN. Thus, the anti-tTG cut-off value can be combined with clinical judgment to diagnose CD, limiting the use of invasive endoscopy.

Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.