Kongensin a attenuates intervertebral disc degeneration by inhibiting TAK1-mediated PANoptosis of nucleus pulposus cells.

Low back pain (LBP) is most commonly caused by intervertebral disc degeneration (IVDD). Pyroptosis, apoptosis, and necroptosis are crucial in IVDD pathogenesis; however, possible simultaneous occurrence in IVDD and co-regulation between the pathways and the regulatory mechanisms have not been investigated. PANoptosis is a regulated cell death (RCD) pathway with the key characteristics of pyroptosis, apoptosis, and necroptosis. This study revealed that tert-butyl hydroperoxide (TBHP) altered the expression of key proteins involved in PANoptosis in nucleus pulposus cells (NPCs). Furthermore, the natural product Kongensin A (KA), which has potential anti-necrotic and anti-inflammatory properties, inhibited PANoptosis. TAK1, often referred to as mitogen-activated protein kinase kinase kinase 7 (Map3k7), is a key regulator of innate immunity, cell death, inflammation, and cellular homeostasis; however, the physiological roles and regulatory mechanisms underlying IVDD remain unclear. In this study, we discovered that KA can upregulate TAK1 expression in NPCs, -which inhibits PANoptosis by suppressing oxidative stress. In conclusion, our results suggest that KA inhibits PANoptosis and delays IVDD progression in NPCs by upregulating TAK1 expression to maintain mitochondrial redox balance. Consequently, targeting TAK1 may be a promising therapeutic approach for IVDD therapy.

PPAR γ activation in chondrocytes alleviates glucocorticoid-induced oxidative stress, mitochondrial impairment, and pyroptosis via autophagic flow enhancement.

Osteoarthritis (OA) is a progressive age-related disease characterised by pathological changes in the synovium, articular cartilage, and subchondral bone, significantly reducing the patients' quality of life. This study investigated the role of glucocorticoids, specifically dexamethasone, in OA progression, with a particular focus on their effects on chondrocytes. Although glucocorticoids are commonly used for OA pain relief, our research demonstrated that high concentrations of dexamethasone may accelerate OA progression by enhancing the ability of reactive oxygen species to inhibit chondrocyte autophagy, resulting in cell death and accelerated cartilage degeneration. Despite reports on the acceleration of pathogenesis and cartilage damage in some patients of OA taking corticosteroids, the mechanism behind the same has not been investigated. This necessitates an investigation of the concentration-dependent changes in the cartilage cells upon dexamethasone administration. In addition, the protective effect of PPAR γ on chondrocytes can prevent the decrease in chondrocyte autophagy and delay cartilage degeneration. Therefore, our study suggests that the therapeutic use of glucocorticoids in OA treatment should be more nuanced considering their potential detrimental effects. Future investigations should focus on the mechanisms underlying the glucocorticoid-mediated modulation of cell death processes, which could provide insights into new therapeutic strategies for OA treatment.

The prediction and treatment of postpartum myofascial pelvic pain.

BACKGROUND: The clinical manifestations of myofascial pelvic pain (MFPP) are mainly acute or chronic muscle pain at one or more trigger points in the pelvic cavity or pelvic floor. OBJECTIVE: This study aims to explore the predictive value of pelvic floor myoelectric parameters with respect to MFPP and the effect of its clinical treatment. METHODS: Two hundred and one women followed up in the Wenzhou People's Hospital 6-12 weeks postpartum between July 2020 and July 2021. They were divided into an MFPP group (n= 90) and a non-MFPP group (n= 102), but 9 MFPP patients without a pelvic floor electromyography evaluation were not included. The general demographic data and pelvic floor electromyography evaluation parameters of the two groups were compared; the related factors of postpartum women suffering from MFPP were analyzed, and a nomogram model of the postpartum risk of suffering from MFPP was established. The 99 patients with postpartum MFPP were divided into a treatment group (n= 10) and a control group (n= 89). The difference in visual analog scale scores between the two groups initially and after three months of treatment was compared to evaluate the effective remission rate of postpartum MFPP after treatment. RESULTS: A significant difference was observed in the relaxation time at the rapid contraction stage (z= 4.369, p< 0.05) and the tension contraction stage (z= 135.645, p< 0.01) between the MFPP group and the non-MFPP group. The nomogram model for predicting postpartum MFPP was established with nine variables as potential predictors. The calibration chart and C index of 0.68 (95% CI: 0.65-0.71) proved that the model had a certain degree of discrimination. The clinical decision-making curve showed that the model could increase the net benefit rate of patients. The pain relief rate in the treatment group was significantly higher than that in the control group (p< 0.01). CONCLUSION: There is a significant correlation between postpartum MFPP and relaxation time at rapid contraction stage and tension contraction stage. The risk prediction nomogram model of postpartum MFPP established with nine potential predictors has a certain prediction capability, and clinical treatment can effectively relieve MFPP in postpartum patients.

Serum RANKL levels in Chinese patients with ankylosing spondylitis: a meta-analysis.

OBJECTIVE: We aimed to determine the association between serum receptor activator of nuclear factor-kappa B ligand (sRANKL) levels and ankylosing spondylitis (AS) in Chinese patients. METHODS: The PubMed, Cochrane Library, Embase, Chinese Biomedical Database, Web of Science, China National Knowledge Infrastructure, VIP, and Wan Fang databases were searched for studies conducted before October 1, 2020, without language restrictions. STATA version 12.0 and Revman version 5.3 were used to analyze the data. The standard mean differences (SMDs) and corresponding 95% confidence intervals (95% CIs) were calculated. RESULTS: Twelve clinical case-control studies, including 585 patients with AS and 423 healthy controls, were included. The combined SMD for sRANKL suggested that the sRANKL level was significantly higher in Chinese patients with AS than in healthy controls (SMD: 3.27, 95% CI 2.11-4.43, P < 0.00001). Serum RANKL-related factor osteoprotegerin (OPG) levels (SMD: 0.86, 95% CI 0.09-1.64, P < 0.03) were lower in the Chinese patients with AS than in healthy controls, and the RANKL/OPG ratio (SMD = 1.05, 95% CI 0.64-1.46, P < 0.00001) in Chinese patients with AS was approximately the same as that of healthy controls. Subgroup analysis indicated that patients from North and South China had higher sRANKL levels than controls; the sRANKL levels of patients from South China were higher in the subgroup with a Bath Ankylosing Spondylitis Functional Index (BASFI) of > 4 than those of patients in other subgroups. In terms of duration, patients with AS for > 8 years had higher sRANKL levels than health controls. Other subgroup analyses were conducted by region, language, source of control, age, and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). In these subgroups, the sRANKL levels were significantly higher in the patients with AS than in healthy controls. The BASFI and BASDAI were sources of heterogeneity. CONCLUSIONS: The sRANKL levels are higher in Chinese patients with AS, especially among those from South China. sRANKL levels may be positively correlated with the pathogenesis of AS among Chinese patients.

Knockdown of NLE1 inhibits development of malignant melanoma in vitro and in vivo NLE1 promotes development of malignant melanoma.

Melanoma, which originates from neural crest derived melanocytes, causes severe pain and even death to numerous patients. Previous studies reported that Notchless Homolog 1 (NLE1) plays an important role in cell proliferation, transcription and signal transduction. However, the clinical significance and biological behavior of NLE1 in melanoma remain a mystery. Thus, the role of NLE1 in melanoma was investigated in vitro and in vivo. The expression of NLE1 in melanoma was elevated and the expression level was positively correlated with lymphatic metastasis and tumor stage. In addition, NLE1 knockdown by shRNA specifically inhibited proliferation, enhanced the apoptotic sensitivity and hindered migration of melanoma cells in vitro. Mice xenograft model further showed that NLE1 knockdown could inhibit the tumor formation of melanoma in vivo. Additionally, the induction of apoptosis of melanoma cells by NLE1 knockdown required the participation of a series of apoptosis-related proteins. Besides, NLE1 can activate the PI3K/AKT signaling pathway. In summary, NLE1 was involved in the development and progression of melanoma, which may be a novel potential target for molecular therapy of melanoma.

IL-18-Mediated SLC7A5 Overexpression Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via the c-MYC Pathway.

Objective: To investigate the role of IL-18 in the regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Methods: To assess whether IL-18 affects the osteogenic differentiation of hBMSCs through the c-MYC/SLC7A5 axis, IL-18 dose-response and time-course experiments were performed to evaluate its impact on osteogenic differentiation. To confirm osteogenic differentiation, alizarin red staining calcium measurement were performed. RT-qPCR and western blotting were used to determine the expression levels of bone-specific markers ALP, RUNX2, and BMP2, as well as those of SLC7A5 and c-MYC. Furthermore, SLC7A5 and c-MYC expression was evaluated via immunofluorescence. To elucidate the roles of SLC7A5 and c-MYC in osteoblast differentiation, cells were transfected with SLC7A5 or c-MYC siRNAs, or treated with the SLC7A5-specific inhibitor JPH203 and c-MYC-specific inhibitor 10058-F4, and the expression of SLC7A5, c-MYC, and bone-specific markers ALP, RUNX2, and BMP2 was assessed. Results: Our results demonstrated that IL-18 increased calcium deposition in hBMSCs, and upregulated the expression of SLC7A5, c-MYC, ALP, RUNX2, and BMP2. Silencing of SLC7A5 or c-MYC using siRNA reduced the expression of ALP, RUNX2, and BMP2, while IL-18 treatment partially reversed the inhibitory effect of siRNA. Similar results were obtained by treating hBMSCs with SLC7A5 and c-MYC specific inhibitors, leading to significant reduction of the osteogenesis effect of IL-18 on hBMSCs. Conclusion: In conclusion, our results indicate that IL-18 promotes the osteogenic differentiation of hBMSCs via the SLC7A5/c-MYC pathway and, therefore, may play an important role in fracture healing. These findings will provide new treatment strategies for delayed fracture healing after splenectomy.

Correlation between osteoarthritis and monocyte chemotactic protein-1 expression: a meta-analysis.

OBJECTIVE: We evaluated the association between monocyte chemotactic protein-1 (MCP-1) and osteoarthritis. METHODS: We searched PubMed, Cochrane Library, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), VIP (Chinese database), and Wan Fang (Chinese database) (before May 10, 2020), with no language limitations. STATA version 12.0 and Revman version 5.3 were used for data analysis. The standard mean difference (SMD) and corresponding 95% confidence intervals (95% CIs) were calculated. Nine clinical studies, including 376 patients with osteoarthritis and 306 healthy controls, were evaluated. RESULTS: The combined SMDs of MCP-1 expression levels suggested that MCP-1 expression was significantly higher in patients with osteoarthritis than healthy controls (SMD = 1.97, 95% CI = 0.66-3.28, p = 0.003). Moreover, subgroup analysis implied that osteoarthritis patients from both Asians and mixed populations had higher MCP-1 expression levels than controls, whereas Caucasians did not (p > 0.05). Serum MCP-1 levels (SMD = 2.83, 95% CI = 1.07-4.6, p < 0.00001) were significantly higher in patients with osteoarthritis than in controls; however, this difference was not significant in synovial fluid and cartilage tissue. Subgroup analysis for ethnicity showed that MCP-1 levels were significantly higher in Chinese, Dutch, and Brazilian patients with osteoarthritis than in control groups, although significant differences were not observed for American and Italian subgroups. CONCLUSIONS: Our meta-analysis demonstrated that MCP-1 expression levels were higher in patients with osteoarthritis than in healthy controls and that MCP-1 may play important roles in the progression of osteoarthritis. Serum MCP-1 levels may serve as a potential biomarker for the diagnosis of osteoarthritis.

STMN1 and MKI67 Are Upregulated in Uterine Leiomyosarcoma and Are Potential Biomarkers for its Diagnosis.

BACKGROUND The aim of this study was to investigate STMN1 and MKI67 expression in uterine leiomyosarcoma and their potential roles as biomarkers for diagnosis. MATERIAL AND METHODS The expression of STMN1 and MKI67 mRNA in uterine leiomyosarcoma were investigated in TCGA database. The overall survival (OS) and disease-free survival (DFS) were compared between high and low expression groups. Seventy-two patients who received hysterectomy were included and divided into 4 groups: uterine normal smooth muscle tissue (UNSM=30), uterine leiomyoma (UL=30), uterine cellular leiomyoma (UCL=24), and uterine leiomyosarcoma (ULS=18). The STMN1 and MKI67 protein expression of the 4 groups were examined by immunohistochemistry (IHC) assay. RESULTS The expression level of STMN1 mRNA in cancer tissue was significantly higher than those of normal uterine smooth muscle tissue. The high and low expression of STMN1 and mki67 gene mRNA was not related to the patients' OS and DFS (P>0.05). The positive rate of STMN1 protein in uterine leiomyosarcoma was 100.00%, which was significantly higher than that of the other 3 groups (χ²=11.72, P=0.008). And the positive rate of KIM67 protein in uterine leiomyosarcoma was 77.78%, which was also significantly higher than that of the other 3 groups (χ²=48.89, P=0.000). The diagnostic sensitivity and specificity were 77.78%, 90.74% for STMN1 combined MKI67 with the positive predictive value and negative predictive value of 73.68% and 92.45%, respectively. CONCLUSIONS STMN1 and MKI67 were upregulated in uterine leiomyosarcoma and act as potential biomarkers for uterine leiomyosarcoma diagnosis.

Roles of two cyp11 genes in sex hormone biosynthesis in Japanese flounder (Paralichthys olivaceus).

The P450 side-chain cleavage enzymes P450scc (Cyp11a) and 11ß-hydroxylase (Cyp11b) play important roles in sex steroid and cortisol production. Here, two duplicates of cyp11 genes were identified in Japanese flounder (Paralichthys olivaceus): Pocyp11a and Pocyp11b, respectively. Phylogenetic analysis and amino acid sequence alignment revealed that Pocyp11a and Pocyp11b shared significant identity with sequences of other teleost fish species. The quantitative real-time polymerase chain reaction (qRT-PCR) results indicated that among the studied tissues, brain tissue showed the highest expression of Pocyp11a, followed by kidney and testis tissues, whereas Pocyp11b expression was highest in the testis. The expression patterns of these two genes showed sexual dimorphism, with both genes showing higher expression in the testis than in the ovary. In-situ hybridization analysis demonstrated that Pocyp11a and Pocyp11b mRNA were both detected in oocytes, spermatocytes, and Sertoli cells, indicating that they might be involved in hormone synthesis. The expression levels of Pocyp11a and Pocyp11b were significantly downregulated by treatment with 17α-methyltestosterone (17α-MT) in the testis and ovary in both in vivo and studies. In vivo studies showed that Pocyp11a and Pocyp11b transcripts were suppressed by 17ß-estradiol (E2 ) treatment in both the testis and ovary. In addition, in vitro studies showed that the expression level of Pocyp11b was decreased by treatment with E2 , whereas that of Pocyp11a was largely unaffected. Moreover, the expression levels of Pocyp11a and Pocyp11b in the testis cell line were significantly upregulated after NR0b1 and NR5a2 (p < .05) treatment. These results indicate that Pocyp11a and Pocyp11b might play important roles in sex hormone biosynthesis. Our research can assist future studies of the mechanisms of steroid biosynthesis and functional differences between cyp11a and cyp11b in Japanese flounder.

Two cyp17 genes perform different functions in the sex hormone biosynthesis and gonadal differentiation in Japanese flounder (Paralichthys olivaceus).

P450c17, a key enzyme in the steroid generation pathway, plays an important role in the production of sex steroid and cortisol. In this study, two cyp17 gene isoforms, Pocyp17-I and Pocyp17-II were isolated from Paralichthys olivaceus gonads. Domain architecture analysis of Pocyp17-I and Pocyp17-II revealed that they had three regions important to enzymatic function. Structural analysis showed that Pocyp17-I and Pocyp17-II had 8 and 9 exons respectively, and the difference was caused by the insertion of an extra intron (intron1) in the latter. Quantitative real-time polymerase chain reaction results indicated that the expression of these two genes showed sexually dimorphism that Pocyp17-I and Pocyp17-II were highest expressed in testis and ovary, respectively. The in situ hybridization analysis of gonads indicated that Pocyp17-I and Pocyp17-II mRNA were both detected in oocytes, spermatocytes and Sertoli cells. After injection of androgen and estrogen (17α-methyltestosterone, 17ß-estradiol) of different concentrations, the expression level of Pocyp17-I decreased significantly (P < 0.01), whereas estrogen had no influence on Pocyp17-II, but androgen upregulated the expression of Pocyp17-II (P < 0.05). Moreover, Pocyp17-I expression level was down-regulated significantly by NR0b1 but up-regulated by NR5a2 (P < 0.05), whereas Pocyp17-II expression level was down-regulated significantly by NR0b1 and NR5a2 (P < 0.05). All these results demonstrated that there were differences in expression patterns, feedback actions of sex hormones and transcriptional regulations between cyp17-I and cyp17-II, which revealed that cyp17-I and cyp17-II might perform different functions in sex hormones biosynthesis and gonadal differentiation in Japanese flounder.

Roles of piwil1 gene in gonad development and gametogenesis in Japanese flounder, Paralichthys olivaceus.

PIWI family member piwil1, which associates with Piwi-interacting RNA (piRNA), is responsible in regulation of germ cell differentiation and maintenance of reproductive stem cells. In this study, we analyzed the piwil1 gene in Paralichthys olivaceus. Bioinformatics analysis and structure prediction showed that piwil1 had the conserved domains: PAZ domain and PIWI domain. Expression analysis during embryonic development implied that piwil1 gene was maternally inherited. The tissue distribution showed a sexually dimorphic gene expression pattern, with higher expression level in testis than ovary. In situ hybridization results demonstrated that piwil1 was predominantly distributed in oogonia, oocytes, sertoli cells and spermatocytes. A CpG island was predicted in the 5'-flanking region of piwil1 gene, and its methylation levels showed significant disparity between males and females, indicating that the sexually dimorphic expression of piwil1 gene might be regulated by methylation. Furthermore, we explored the distinct roles of human chorionic gonadotropin and 17α-methyltestosterone in regulating the expression of piwil1, and found that piwil1 was interacting with the HPG axis hormones. These results indicated that piwil1 might play a crucial role in gonadal development and gametogenesis in Paralichthys olivaceus.

The evolution and possible role of two Sox8 genes during sex differentiation in Japanese flounder (Paralichthys olivaceus).

Sox8 genes, as members of the Sox family, have been studied widely in mammals. However, regulation of sox8 genes in teleosts has rarely been studied, and functional analysis of these genes in teleosts has rarely been performed. Here, two duplicates of sox8 genes were identified in Japanese flounder, Posox8a and Posox8b. The analysis of expression showed that Posox8a and Posox8b were expressed in Sertoli cells of the testis, indicating that they play important roles in development and functional maintenance of the testis. Positive selection and phylogenetic analysis found that both Posox8a and Posox8b underwent the purification selection during evolutionary and that sox8 was most likely to be the ancestor sox8a. These results suggested that both Posox8a and Posox8b had important biological functions after generation from three rounds of whole-genome duplication in Japanese flounder. The functional differentiation of Posox8a and Posox8b was verified using cell transfection and dual-luciferase reporter assays; Posox8a overexpression-promoted 3ß-hydroxysteroid dehydrogenase expression and Posox8b overexpression-promoted cytochrome P450 aromatase (cyp19a1; P450arom) expression. Finally, combined with Posox8a and Posox8b expression analysis from 30 to 100 days after hatch, we speculated that Posox8a and Posox8b might participate in the process of sex differentiation and gonadogenesis by regulating sex hormone biosynthesis in the Japanese flounder. Our study is the first to demonstrate the possible mechanism of Posox8a and Posox8b in Japanese flounder sex differentiation and gonadogenesis, laying a solid foundation for functional studies of sox8 genes in teleosts.

[Treatment of hepatic cancer in mice by beta-elemene combined DC/Dribble vaccine: an immune mechanism research].

OBJECTIVE: To observe the therapeutic effects of beta-elemene combined DC/Dribble vaccine in treating mice with hepatic cancer, thus exploring their anti-tumor mechanisms. METHODS: Dentritic cells were derived from Balb/c mice's spleen and their phenotypes were identified. Using hepatic cancer cell line BNL1MEA.7R.1 (abbreviated as BNL) originated from Balb/c mice as target cell, DC/Dribble vaccine was prepared via raising the antigen representing carrier autophagosomes (DRips in Blebs, DRibbles), which were rich in tumor antigen information. The mice previously immunized were divided into 4 groups, i.e., the control group, the beta-elemene group, the vaccine group, and the combined group. The PBS was subcutaneously and intraperitoneally injected to mice in the control group. The beta-elemene was intraperitoneally injected at the daily dose of 50 mg/kg to mice in the beta-elemene group and the combined group for 7 successive days. DC/Dribble vaccine was injected into the lymph node of mice in the vaccine group and the combined group on the 1st day, and DC/Dribble vaccine was subcutaneously injected on the 3rd day and the 5th day. All the mice were sacrificed on the 10th day. Their spleens were obtained sterilely, and the suspension was incubated with or without Dribble. The cells were inoculated for 72 h. The contents of IFN-gamma in the supernatant were measured by ELISA. In addition, the spleen cells obtained from the combined group were incubated with different stimulations for 72 h, which were then divided into the control group, the DRibble group, the DC group, and the DC/Dribble vaccine group. The supernatant of cultured cells were collected and the contents of IFN-gamma were measured by ELISA. The liver tumor-bearing mouse model was established, and then the BNL bearing mice were randomly divided into 4 groups, i.e., the control group, the beta-elemene group, the vaccine group, and the combined group. The treatment ways were the same as the immune ways. The tumor size and the survival period were observed in each group. On the 23rd day the mice were sacrificed. The tumor tissue was stripped and stained by HE staining. The pathomorphological manifestations of the tumor tissue were observed by light microscope. RESULTS: In vitro detection of mice immunized previously by different ways showed that the secretion of IFN-gamma was significantly higher in the combined group than in the rest groups (P < 0.01). The secretion of IFN-gamma was significantly higher in the beta-elemene group and the vaccine group than in the control group (P < 0.01). The spleen cells could be stimulated to secrete a large amount of IFN-gamma in the vaccine group and the Dribble group (P < 0.01). When the beta-elemene was 10 microg/mL as the stimulating dose, the secretion of IFN-gamma obviously increased (P < 0.01). In vivo observation showed that the growth velocity of tumors in mice of the combined group was slowed down. There was statistical difference in the tumor area or the survival period of mice in the combined group, when compared with the other groups (P < 0.01). In HE staining, the surrounding connective tissues of the tumor were wrapped tightly and compactedly, with infiltration of a large amount of inflammatory cells. CONCLUSIONS: beta-elemene combined DC/Dribble vaccine could induce specific immune cells to secrete secretory cells, thus exerting its anti-tumor effect. Its immunological effects might be associated with enhancing the DC antigen presenting function.

[Effects of qingchang huashi recipe on the dendritic cells of the colonic mucosa and the mesenteric lymph nodes in experimental colitis rats].

OBJECTIVE: To observe the effects of qingchang huashi recipe (QHR) on the dendritic cells (DCs) of experimental colitis rats, thus exploring its possible mechanisms for treating ulcerative colitis (UC). METHODS: The UC rat model was induced by TNBS/anhydrous alcohol. Forty male Wistar rats were randomly divided into 4 groups, i.e., the normal group, the model group, the QHR group, and the Mesalazine group, 10 in each group. Since the 2nd day of modeling, corresponding medication was respectively administered to each treatment group by gastrogavage for 10 successive days. The number of DCs in the colonic mucosa was observed using iMmunohistochemical assay. The DCs ratio in the mesenteric lymph nodes, and the expressions of surface molecules MHC-II and CD86 were detected using flow cytometry. RESULTS: Compared with the model group, the number of DCs in the colonic mucosa significantly decreased, the expression of MHC-II in the mesenteric lymph nodes significantly decreased in the QHR group and the Mesalazine group, showing statistical difference (P < 0.01). There was no statistical difference between the two groups (P > 0.05). There was no statistical difference in the DCs ratios and the CD86 expression among the 4 groups (P > 0.05). CONCLUSION: QHR could decrease the infiltration of DCs in the colonic mucosa, and suppress the activation of DCs in the mesenteric lymph nodes, which might be one of its mechanisms for treating UC.