BACKGROUND: Fibrosis of the connective tissue in the vaginal wall predominates in pelvic organ prolapse (POP), which is characterized by excessive fibroblast-to-myofibroblast differentiation and abnormal deposition of the extracellular matrix (ECM). Our study aimed to investigate the effect of ECM stiffness on vaginal fibroblasts and to explore the role of methyltransferase 3 (METTL3) in the development of POP. METHODS: Polyacrylamide hydrogels were applied to create an ECM microenvironment with variable stiffness to evaluate the effects of ECM stiffness on the proliferation, differentiation, and expression of ECM components in vaginal fibroblasts. METTL3 small interfering RNA and an overexpression vector were transfected into vaginal fibroblasts to evaluate the effects of METTL3 silencing and overexpression on matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the ECM. Both procedures were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, Western blotting (WB), quantitative real-time polymerase chain reaction (RT-qPCR), and immunofluorescence (IF). RESULTS: Vaginal fibroblasts from POP patients exhibited increased proliferation ability, increased expression of α-smooth muscle actin (α-SMA), decreased expression of collagen I/III, and significantly decreased expression of tissue inhibitors of matrix metalloproteinases (TIMPs) in the stiff matrix ( P <0.05). Compared with those from non-POP patients, vaginal wall tissues from POP patients demonstrated a significant increase in METTL3 content ( P <0.05). However, silencing METTL3 expression in vaginal fibroblasts with high ECM stiffness resulted in decreased proliferation ability, decreased α-SMA expression, an increased ratio of collagen I/III, and increased TIMP1 and TIMP2 expression. Conversely, METTL3 overexpression significantly promoted the process of increased proliferation ability, increased α-SMA expression, decreased ratio of collagen I/III and decreased TIMP1 and TIMP2 expression in the soft matrix ( P <0.05). CONCLUSIONS Elevated ECM stiffness can promote excessive proliferation, differentiation, and abnormal ECM modulation, and the expression of METTL3 plays an important role in alleviating or aggravating matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal ECM modulation.
Humans ; Female ; Extracellular Matrix/metabolism* ; Cell Differentiation/genetics* ; Methyltransferases/metabolism* ; Pelvic Organ Prolapse/pathology* ; Fibroblasts/metabolism* ; Myofibroblasts/metabolism* ; Vagina/metabolism* ; Cell Proliferation/physiology* ; Cells, Cultured ; Middle Aged

BackgroundNarrative exposure therapy （NET）， an integration of narrative therapy and exposure therapy， has been shown to be effective in relieving the symptoms of post-traumatic stress disorder （PTSD）， which can help patients gain a deeper understanding of their trauma and is also considered to be quite safe. PTSD is highly prevalent in children and adolescents， while the effectiveness of NET intervention varies among the subjects. ObjectiveTo systematically evaluate the effectiveness of NET for PTSD in children and adolescents， so as to provide references for the clinical application of NET. MethodsOn August 1， 2022， the Cochrane Library， PubMed， Web of Science， CINAHL， China National Knowledge Infrastructure （CNKI）， SinoMed， VIP and Wanfang database were searched from their inception to June 2022. Search was conducted with the use of a combination of medical subject heading and free text terms， and randomized controlled trials relevant to NET for PTSD in children and adolescents were collected. Then the quality of the controlled trials was evaluated according to the Cochrane Collaboration's tool for assessing risk of bias （2011）， and Meta-analysis was performed using RevMan 5.4 software. ResultsNine randomized controlled trials involving 394 children and adolescents with PTSD were included. Meta-analysis showed that NET and relaxation therapy reported comparable symptom relief in PTSD patients within 1 to 3 months after intervention （SMD=0.22， 95% CI： -0.84~1.28） and at 6 months after intervention （SMD=0.21， 95% CI： -0.75~1.17）， while NET provided greater PTSD symptom relief than routine therapy both within 1 to 3 months after intervention （SMD=-0.66， 95% CI： -1.04~-0.27） and at 6 months after intervention （SMD=-0.77， 95% CI： -1.36~-0.19）， with statistically significant differences. Regarding the alleviation of depressive symptoms， the effect was similar between NET and routine therapy within 1 to 3 months after intervention （SMD=-0.39， 95% CI： -0.98~0.21） and at 6 months after intervention （SMD=-0.74， 95% CI： -2.23~0.75）. No statistical difference was demonstrated between NET and routine therapy in relieving psychological distress （SMD=-0.54， 95% CI： -2.14~1.07） and suppressing hyperorexia （SMD=-0.17， 95% CI： -0.54~0.19） 1 to 3 months after intervention. ConclusionNET yields a better outcome and a medium- and long-term effectiveness in alleviating symptoms of PTSD in children and adolescents compared with routine therapy， while it does not offer any significant advantages in improving depression symptoms， psychological distress and hyperorexia.

Objective:To preliminarily investigate the applicability of a poliovirus pseudovirus-based neutralization assay in evaluating the immunogenicity of recombinant poliovirus vaccines and their in vivo potency in rats. Methods:Serum samples from rats immunized with recombinant poliovirus vaccines were tested using both the pseudovirus neutralization assay and the live-virus neutralization assay with Sabin strain. The consistency and correlation of the two methods were analyzed using the Kappa test and Spearman′s rank correlation.Results:For the neutralizing antibodies against typeⅠ, Ⅱ, and Ⅲ polioviruses, the Kappa values for consistency analysis of the two methods were 0.914, 1.000, and 0.751, respectively ( P<0.001), and the correlation coefficients ( R values) were 0.833, 0.927, and 0.859, respectively ( P<0.001). Conclusions:The test results of the two methods are consistent and show a good correlation, indicating that the pseudovirus neutralization assay can be applied to evaluating the immunogenicity of poliovirus vaccines and also can be used in rat potency tests.

Background Flurochloridone (FLC) is toxic to male reproduction and can induce apoptosis of testicular tissue and supporting cells under oxidative stress. Of particular concern is whether nuclear factor-erythrocyte 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway and nuclear factor kappa B (NFκB) signaling pathway participate this process. Objective To observe apoptosis of testicular tissue and sertoli TM4 cells and alterations of Nrf2/HO-1 and NFκB signaling pathways in mice treated with FLC in vivo/in vitro. Methods (1) Animal experiment. Testis samples were harvested from male C57BL/6 mice after 28-day FLC (0, 3, 15, 75, and 375 mg·kg⁻¹ per day) exposure via oral route. Malondialdehyde (MDA) and superoxide dismutase (SOD) in homogenate of testicular tissue were measured by colorimetry. Apoptosis of testicular tissue was evaluated by TUNEL staining. Expression and distribution of Nrf2 and NFκB were detected by immunohistochemistry. Protein expression levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), NFκB, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), and phosphorylated recombinant inhibitory subunit of nuclear factor kappa-B alpha (P-IκBα) in testicular tissue homogenate were determined by Western blotting. (2) Cell experiment. TM4 cell lines were treated with 40, 80, 120, 160, and 200 μmol·L⁻¹ FLC for 6 h, and cell viability was detected by CCK-8. After 6 h exposure to 40, 80, and 160 μmol·L⁻¹ FLC, the apoptosis rate was detected by flow cytometry, and the protein expression levels of Nrf2, HO-1, NQO1, NFκB, IKKβ, and IκBα were detected by Western blotting. Results (1) Animal experiment. Apoptosis occurred in the interstitial and basal parts of spermatogenic tubules in male C57BL/6 mice after 28 days of oral FLC exposure. Compared with the control group, the MDA level in testicular tissue of the 375 mg·kg⁻¹ FLC-treated group was significantly increased (P<0.05), and the SOD activity was significantly decreased (P<0.05). After 375 mg·kg⁻¹ FLC exposure, apoptosis occurred in the interstitial and basal parts of spermatogenic tubules. The results of immunohistochemistry showed the expression of Nrf2 and NFκB in the interstitium and basal part of spermatogenic tubules of the treated groups. Compared with the control group, the protein levels of Nrf2, NQO1, P-IκBα, NFκB, and IKKβ in the 15, 75, and 375 mg·kg^-1 groups were significantly increased (P<0.001), and the HO-1 protein level was significantly increased in the 375 mg·kg⁻¹ group (P<0.001). (2) Cell experiment. Compared with the control group, the TM4 cell viabilities in the 40, 80, 120, 160, and 200 μmol·L⁻¹ FLC-treated groups significantly decreased (P<0.01). The apoptosis rates were significantly increased (P<0.05), and the apoptosis rates increased from 5.7% in the control group to 7.4%, 9.4%, and 11.7% in the 40, 80, and 160 μmol·L⁻¹, respectively. The Nrf2 protein level in the 40 μmol·L⁻¹ group was significantly increased (P<0.01), while the levels significantly decreased in the 80 and 160 μmol·L⁻¹ groups (P<0.01). The HO-1 protein levels in the 40, 80, and 160 μmol·L⁻¹ groups were significantly increased (P<0.01). The level of NQO1 protein in the 40 μmol·L⁻¹ group was significantly increased (P<0.01). The NFκB protein levels were significantly increased in the 80 and 160 μmol·L⁻¹ groups (P<0.001). The IκBα protein levels were significantly decreased in all treated groups (P<0.001). The IKKβ protein had no significant change. Conclusion FLC induces testicular tissue apoptosis, and the process affects Nrf2/HO-1 signaling pathway and NFκB signaling pathway. The in vitro study confirms that FLC could induce apoptosis of TM4 cells and activate Nrf2/HO-1 and NFκB signaling pathways.

Background Flurochloridone (FLC) can induce apoptosis in Sertoli cells, but the specific mechanism remains unknown. Objective To investigate the testicular cell apoptosis in mice as well as apoptosis and activation of endoplasmic reticulum stress in TM4 cell line induced by FLC through in vivo and in vitro study designs respectively, and study the role of inosital-requiring enzyme 1α (IRE1α)-c-Jun N-terminal kinase (JNK) signaling pathway in the process of FLC-induced apoptosis in TM4 cells through intervention study design. Methods Testicular tissues were collected from male C57BL/6 mice which were treated with 3, 15, 75, and 375 mg·（kg·d）⁻¹ FLC by oral perfusion for 28 d. Apoptosis was observed by TUNEL staining, and the levels of apoptosis-related proteins were detected by Western blotting, including B-cell lymphoma-2 (Bcl-2), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2 associated X protein (Bax). In the in vitro study, TM4 cells were treated with different concentrations of FLC (40, 80, and 160 μmol·L⁻¹) for 6 h, then apoptosis rate was detected by flow cytometry, and the levels of apoptosis-related proteins (Bcl-2, Bim, and Bax) and endoplasmic reticulum stress-related proteins [glucose regulated protein 78 (GRP78), phosphorylated-protein kinase R like endoplasmic reticulum kinase (p-PERK), activating transcription factor 6 (ATF6), phosphorylated-inosital-requiring enzyme 1α (p-IRE1α), and phosphorylated-JNK (p-JNK)] were measured by Western blotting. In the intervention study, TM4 cells were pretreated with IRE1α phosphorylation inhibitor 4μ8C and JNK phosphorylation inhibitor SP600125 for 6 h, then treated with 160 μmol·L⁻¹ FLC for 6 h. The levels of apoptosis-related proteins and endoplasmic reticulum stress-related proteins were measured by Western blotting, and cell viability was detected by cell counting kit-8. Results After the male C57BL/6 mice orally exposed to FLC for 28 d, apoptosis occurred in the seminiferous tubule. The protein expression level of Bcl-2, apoptosis inhibitor, was decreased in the 75 and 375 mg·(kg·d)⁻¹ groups (P<0.05), and the protein expression levels of Bim and Bax, apoptosis promoters, were increased in the 75 and 375 mg·(kg·d)⁻¹ groups respectively (P<0.05). The percentages of apoptotic cells in the 0, 40, 80, and 160 μmol·L⁻¹ FLC groups were 2.7%±0.2%, 4.8%±1.3%, 9.4%±0.3%, and 13.2%±0.2%, respectively, increased significantly compared with the control group (P<0.05). The protein expression level of Bcl-2 also was decreased in the 160 μmol·L⁻¹ FLC group (P<0.05), while the levels of Bim and Bax were increased in both of the 80 and 160 μmol·L⁻¹ groups (P<0.05). The expression levels of endoplasmic reticulum stress-related proteins (GRP78, p-PERK, ATF6, p-IRE1α, and p-JNK) were increased (P<0.05) or showed a rising trend in TM4 cells. Pre-treatment with 4μ8C (25 and 50 μmol·L⁻¹) and SP600125 (10 and 20 μmol·L⁻¹) significantly down-regulated the protein expression levels of GRP78, p-IRE1α, p-JNK, and Bax induced by FLC (P<0.05) or in a downward trend. Both of the inhibitors alleviated the decreased cell viability induced by FLC (P<0.05) or in alleviating fashion. Conclusion FLC could induce apoptosis in mice testis and TM4 cell apoptosis through activating endoplasmic reticulum stress and IRE1α-JNK signaling pathway.

Hypervirulent Klebsiella pneumoniae, short for hvKP, is a hypervirulent variant of classical Klebsiella pneumoniae, which accounts for serious infection in healthy people, exhibits strong pathogenicity, high mortality and poor prognosis. At present, hvkp is of high prevalence all over the world, and the infection rate shows a continuous upward trend, which brings great challenges to public health security and clinical treatment. This paper summarized the research progress on virulence factors of hvkp, such as capsular polysaccharides, siderophore, lipopolysaccharide, adhesins and recently discovered Type Ⅵ secreting system, and aimed to deepen the understanding and recognition of hvKP.

Hypervirulent Klebsiella pneumoniae, short for hvKP, is a hypervirulent variant of classical Klebsiella pneumoniae, which accounts for serious infection in healthy people, exhibits strong pathogenicity, high mortality and poor prognosis. At present, hvkp is of high prevalence all over the world, and the infection rate shows a continuous upward trend, which brings great challenges to public health security and clinical treatment. This paper summarized the research progress on virulence factors of hvkp, such as capsular polysaccharides, siderophore, lipopolysaccharide, adhesins and recently discovered Type Ⅵ secreting system, and aimed to deepen the understanding and recognition of hvKP.

Pseudomonas aeruginosa is one of the common multidrug-resistant bacteria in the clinic. Because it can produce a "protective" biofilm, it can affect the penetration and killing efficacy of antibacterial drugs, leading to the formation of a persistent and persistent chronic infection in the host. Biofilms make Pseudomonas aeruginosa resistant to antibacterials and evasive to the host′s immune system. Therefore, traditional conventional antibacterials are difficult to achieve effective bactericidal treatment. Understanding the process of P. aeruginosa biofilm formation and the regulatory mechanisms that affect biofilms can provide ideas and methods for our future research on new antibacterial drugs.

Pseudomonas aeruginosa is one of the common multidrug-resistant bacteria in the clinic. Because it can produce a "protective" biofilm, it can affect the penetration and killing efficacy of antibacterial drugs, leading to the formation of a persistent and persistent chronic infection in the host. Biofilms make Pseudomonas aeruginosa resistant to antibacterials and evasive to the host′s immune system. Therefore, traditional conventional antibacterials are difficult to achieve effective bactericidal treatment. Understanding the process of P. aeruginosa biofilm formation and the regulatory mechanisms that affect biofilms can provide ideas and methods for our future research on new antibacterial drugs.

Objective To explore the influence of apoptosis-associated speck-like protein containing a caspase recruitment do-main(ASC)and Caspase-1 on the pathogenesis of primary biliary cirrhosis(PBC).Methods The real-time PCR,Western blot,py-rophosphate sequencing and ELISA were adopted to respectively detect the relative expressions of mRNA and protein of peripheral blood mononuclear cells(PBMS)Caspase-1 and ASC as well as the methylation status of ASC promoter region and plasma Caspase-1 and IL-18 expression levels in 30 cases of PBC and healthy controls.Results The mRNA and protein expressions of PBMC Caspase-1 and ASC in the PBC group were significantly higher than those in the control group(P<0.05).The methylation rate of ASC promoter Island1(ISI)was significantly lower than that of the control group(P<0.05),which of Island 2(IS2)was smaller than the background value and had no methylation occurrence.The levels of plasma Caspase-1 and IL-18 in the PBC group were sig-nificantly higher than those in the control group(P<0.05).The ASC mRNA in the PBC group was significantly correlated with the Caspase-1 mRNA expression(P<0.05);the methylation rates at loci 1,2,4,5 of ASC promoter region CpG island were nega-tively correlated with ASC mRNA expression(P< 0.05),and which at loci 3,6 had no correlation with their expressions(P>0.05);plasma Caspase-1 and IL-18 levels showed the obviously positive correlation.Conclusion ASC and Caspase-1 are involved in the immune inflammatory response in PBC.