Temporomandibular joint osteoarthritis (TMJ-OA) is a common disease often accompanied by pain, seriously affecting physical and mental health of patients. Abnormal innervation at the osteochondral junction has been considered as a predominant origin of arthralgia, while the specific mechanism mediating pain remains unclear. To investigate the underlying mechanism of TMJ-OA pain, an abnormal joint loading model was used to induce TMJ-OA pain. We found that during the development of TMJ-OA, the increased innervation of sympathetic nerve of subchondral bone precedes that of sensory nerves. Furthermore, these two types of nerves are spatially closely associated. Additionally, it was discovered that activation of sympathetic neural signals promotes osteoarthritic pain in mice, whereas blocking these signals effectively alleviates pain. In vitro experiments also confirmed that norepinephrine released by sympathetic neurons promotes the activation and axonal growth of sensory neurons. Moreover, we also discovered that through releasing norepinephrine, regional sympathetic nerves of subchondral bone were found to regulate growth and activation of local sensory nerves synergistically with other pain regulators. This study identified the role of regional sympathetic nerves in mediating pain in TMJ-OA. It sheds light on a new mechanism of abnormal innervation at the osteochondral junction and the regional crosstalk between peripheral nerves, providing a potential target for treating TMJ-OA pain.
Animals ; Osteoarthritis/physiopathology* ; Mice ; Sympathetic Nervous System/physiopathology* ; Temporomandibular Joint Disorders/physiopathology* ; Arthralgia ; Sensory Receptor Cells ; Disease Models, Animal ; Norepinephrine ; Male ; Temporomandibular Joint/physiopathology* ; Pain Measurement

OBJECTIVES: To explore the mechanism of Lacticaseibacillus paracasei E6 for improving vinorelbine-induced immunosuppression in zebrafish. METHODS: The intestinal colonization of L. paracasei E6 labeled by fluorescein isothiocyanate (FITC) in zebrafish was observed under fluorescence microscope. In a zebrafish model of vinorelbine-induced immunosuppression, the immunomodulatory activity of L. paracasei E6 was assessed by analyzing macrophage and neutrophil counts in the caudal hematopoietic tissue (CHT), the number of T-lymphocyte, and the expressions of interleukin-12 (IL-12) and interferon-γ (IFN-γ). The contents of short-chain fatty acids (SCFAs) in L. paracasei E6 fermentation supernatant and the metabolites of L. paracasei E6 in zebrafish were detected by LC-MS/MS-based targeted metabolomics. The immunomodulatory effects of the SCFAs including sodium acetate, sodium propionate and sodium butyrate were evaluated in the zebrafish model of immunosuppression. RESULTS: After inoculation, green fluorescence of FITC-labeled L. paracasei E6 was clearly observed in the intestinal ball, midgut and posterior gut regions of zebrafish. In the immunocompromised zebrafish model, L. paracasei E6 significantly alleviated the reduction of macrophage and neutrophil counts in the CHT, increased the fluorescence intensity of T-lymphocytes, and promoted the expressions of IL-12 and IFN-γ. Compared with MRS medium, L. paracasei E6 fermentation supernatant showed significantly higher levels of acetic acid, propionic acid and butyric acid, which were also detected in immunocompromised zebrafish following treatment with L. paracasei E6. Treatment of the zebrafish model with sodium acetate and sodium propionate significantly increased macrophage and neutrophil counts in the CHT and effectively inhibited vinorelbine-induced reduction of thymus T cells. CONCLUSIONS L. paracasei E6 can improve vinorelbine-induced immunosuppression in zebrafish through its SCFA metabolites acetic acid and propionic acid.
Animals ; Zebrafish/immunology* ; Acetic Acid/metabolism* ; Propionates/metabolism* ; Fatty Acids, Volatile/metabolism*

OBJECTIVES: To investigate the efficacy of Lactobacillus plantarum ZG03 (L. plantarum ZG03) for ameliorating oxidative stress in zebrafish. METHODS: We evaluated the growth pattern of L. plantarum ZG03, observed its morphology using field emission scanning electron microscopy, and assessed its safety and potential efficacy with whole-genome sequencing for genetic analysis. FITC-labeled ZG03 was used to observe its intestinal colonization in zebrafish. In a zebrafish model of 2% glucose-induced oxidative stress, the effect of ZG03 was evaluated by assessing the changes in neutrophils in the caudal hematopoietic tissue (CHT), superoxide dismutase (SOD) activity, reactive oxygen species (ROS) levels, and malondialdehyde (MDA) content. Liquid chromatography-mass spectrometry-based targeted metabolomics was used for analyzing short-chain fatty acids (SCFAs) in the zebrafish, and the antioxidant effects of the key metabolites (acetate, propionate, and caproate) were tested. RESULTS: On MRS agar, L. plantarum ZG03 formed circular, smooth, moist, and milky-white colonies with a rod-shaped cell morphology. Genomic analysis revealed abundant sugar metabolism gene clusters. After inoculation of FITC-labeled L. plantarum ZG03 in zebrafish, green fluorescence was clearly observed in the intestinal bulb, mid-intestine, and hind intestine. In zebrafish with glucose-induced oxidative stress, L. plantarum ZG03 significantly reduced ROS levels and the number of neutrophils in the CHT with increased SOD activity. L.plantarum ZG03 significantly increased the content of SCFAs including acetic acid, propionic acid, and caproic acid in zebrafish metabolites. In addition, sodium acetate, sodium propionate, and sodium caproate in the SCFAs significantly increased SOD activity in the zebrafish models. CONCLUSIONS L. plantarum ZG03 ameliorates oxidative stress in a glucose-induced zebrafish model through its metabolites, particularly the SCFAs including acetic acid, propionic acid and caproic acid.
Animals ; Zebrafish/metabolism* ; Oxidative Stress ; Lactobacillus plantarum/metabolism* ; Fatty Acids, Volatile/metabolism* ; Probiotics ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism*

Objective To explore the mechanism of Lacticaseibacillus paracasei E6 for improving vinorelbine-induced immunosuppression in zebrafish.Methods The intestinal colonization of L.paracasei E6 labeled by fluorescein isothiocyanate(FITC)in zebrafish was observed under fluorescence microscope.In a zebrafish model of vinorelbine-induced immunosuppression,the immunomodulatory activity of L.paracasei E6 was assessed by analyzing macrophage and neutrophil counts in the caudal hematopoietic tissue(CHT),the number of T-lymphocyte,and the expressions of interleukin-12(IL-12)and interferon-γ(IFN-γ).The contents of short-chain fatty acids(SCFAs)in L.paracasei E6 fermentation supernatant and the metabolites of L.paracasei E6 in zebrafish were detected by LC-MS/MS-based targeted metabolomics.The immunomodulatory effects of the SCFAs including sodium acetate,sodium propionate and sodium butyrate were evaluated in the zebrafish model of immunosuppression.Results After inoculation,green fluorescence of FITC-labeled L.paracasei E6 was clearly observed in the intestinal ball,midgut and posterior gut regions of zebrafish.In the immunocompromised zebrafish model,L.paracasei E6 significantly alleviated the reduction of macrophage and neutrophil counts in the CHT,increased the fluorescence intensity of T-lymphocytes,and promoted the expressions of IL-12 and IFN-γ.Compared with MRS medium,L.paracasei E6 fermentation supernatant showed significantly higher levels of acetic acid,propionic acid and butyric acid,which were also detected in immunocompromised zebrafish following treatment with L.paracasei E6.Treatment of the zebrafish model with sodium acetate and sodium propionate significantly increased macrophage and neutrophil counts in the CHT and effectively inhibited vinorelbine-induced reduction of thymus T cells.Conclusion L.paracasei E6 can improve vinorelbine-induced immunosuppression in zebrafish through its SCFA metabolites acetic acid and propionic acid.

Objective To investigate the efficacy of Lactobacillus plantarum ZG03(L.plantarum ZG03)for ameliorating oxidative stress in zebrafish.Methods We evaluated the growth pattern of L.plantarum ZG03,observed its morphology using field emission scanning electron microscopy,and assessed its safety and potential efficacy with whole-genome sequencing for genetic analysis.FITC-labeled ZG03 was used to observe its intestinal colonization in zebrafish.In a zebrafish model of 2%glucose-induced oxidative stress,the effect of ZG03 was evaluated by assessing the changes in neutrophils in the caudal hematopoietic tissue(CHT),superoxide dismutase(SOD)activity,reactive oxygen species(ROS)levels,and malondialdehyde(MDA)content.Liquid chromatography-mass spectrometry-based targeted metabolomics was used for analyzing short-chain fatty acids(SCFAs)in the zebrafish,and the antioxidant effects of the key metabolites(acetate,propionate,and caproate)were tested.Results On MRS agar,L.plantarum ZG03 formed circular,smooth,moist,and milky-white colonies with a rod-shaped cell morphology.Genomic analysis revealed abundant sugar metabolism gene clusters.After inoculation of FITC-labeled L.plantarum ZG03 in zebrafish,green fluorescence was clearly observed in the intestinal bulb,mid-intestine,and hind intestine.In zebrafish with glucose-induced oxidative stress,L.plantarum ZG03 significantly reduced ROS levels and the number of neutrophils in the CHT with increased SOD activity.L.plantarum ZG03 significantly increased the content of SCFAs including acetic acid,propionic acid,and caproic acid in zebrafish metabolites.In addition,sodium acetate,sodium propionate,and sodium caproate in the SCFAs significantly increased SOD activity in the zebrafish models.Conclusion L.plantarum ZG03 ameliorates oxidative stress in a glucose-induced zebrafish model through its metabolites,particularly the SCFAs including acetic acid,propionic acid and caproic acid.

Objective:To investigate the effects of oleuropein (OLE) on the migration, invasion, and chemotherapy sensitivity of cervical cancer cells by regulating the phosphatidylinositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathway.Methods:SiHa cells were treated with 0-80 μ mol/L OLEs to detect cell survival rate and screen for the optimal drug concentration. SiHa cells were randomly separated into SiHa group, OLE low concentration (OLE-L) group, OLE medium concentration (OLE-M) group, OLE high concentration (OLE-H) group, and OLE-H+PI3K activator 740Y-P (OLE-H+740Y-P) group. CCK-8 method was applied to detect the proliferation activity of cells in each group. Transwell experiment was applied to detect the migration and invasion abilities of cells in each group. Flow cytometry was applied to detect the apoptosis rate of cells in each group. Western blot was used to detect the expression levels of PI3K-AKT signaling pathway related proteins, epithelial mesenchymal transition (EMT), and resistance related proteins such as epithelial cadherin, vimentin, N-cadherin, and P-glycoprotein (P-gp) of cells in each group.Results:OLEs with concentrations of 5, 10, and 20 μmol/L were selected for subsequent experiments. Compared with the SiHa group, the survival rate, numbers of clone formation, migration and invasion of cells, and the expression levels of p-PI3K/PI3K, p-AKT/AKT, Vimentin, N-cadherin and P-gp in the OLE-L, OLE-M, and OLE-H groups gradually decreased ( P<0.05), while the apoptosis rate and E-cadherin expression level increased ( P<0.05). Compared to that before cisplatin treatment, the apoptosis rate of SiHa cells increased after cisplatin treatment ( P<0.05). The addition of PI3K activator 740Y-P on the basis of high concentration OLE reversed the trend of changes in the above indicators ( P<0.05) . Conclusions:OLE can inhibit the migration and invasion of cervical cancer cells, and improve their chemotherapy sensitivity. Its mechanism of action may be related to the inhibition of the PI3K-AKT signaling pathway.

Objective:To investigate the effects of oleuropein (OLE) on the migration, invasion, and chemotherapy sensitivity of cervical cancer cells by regulating the phosphatidylinositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathway.Methods:SiHa cells were treated with 0-80 μ mol/L OLEs to detect cell survival rate and screen for the optimal drug concentration. SiHa cells were randomly separated into SiHa group, OLE low concentration (OLE-L) group, OLE medium concentration (OLE-M) group, OLE high concentration (OLE-H) group, and OLE-H+PI3K activator 740Y-P (OLE-H+740Y-P) group. CCK-8 method was applied to detect the proliferation activity of cells in each group. Transwell experiment was applied to detect the migration and invasion abilities of cells in each group. Flow cytometry was applied to detect the apoptosis rate of cells in each group. Western blot was used to detect the expression levels of PI3K-AKT signaling pathway related proteins, epithelial mesenchymal transition (EMT), and resistance related proteins such as epithelial cadherin, vimentin, N-cadherin, and P-glycoprotein (P-gp) of cells in each group.Results:OLEs with concentrations of 5, 10, and 20 μmol/L were selected for subsequent experiments. Compared with the SiHa group, the survival rate, numbers of clone formation, migration and invasion of cells, and the expression levels of p-PI3K/PI3K, p-AKT/AKT, Vimentin, N-cadherin and P-gp in the OLE-L, OLE-M, and OLE-H groups gradually decreased ( P<0.05), while the apoptosis rate and E-cadherin expression level increased ( P<0.05). Compared to that before cisplatin treatment, the apoptosis rate of SiHa cells increased after cisplatin treatment ( P<0.05). The addition of PI3K activator 740Y-P on the basis of high concentration OLE reversed the trend of changes in the above indicators ( P<0.05) . Conclusions:OLE can inhibit the migration and invasion of cervical cancer cells, and improve their chemotherapy sensitivity. Its mechanism of action may be related to the inhibition of the PI3K-AKT signaling pathway.

Objective To explore the correlation of contrast-enhanced ultrasound(CEUS)parameters of adenomyoma before and after MR-guided focused ultrasound surgery(MRgFUS)with the therapeutic efficacy.Methods Uterine ultrasound and CEUS data of 26 patients with adenomyoma before and 24 h,1 and 6 months after MRgFUS,as well as MRI before and immediately after MRgFUS were retrospectively analyzed.The lesion volume shown on CEUS and MRI before MRgFUS,the non perfusion volume(NPV)of adenomyoma on MRI immediately after and CEUS 24 h after MRgFUS were compared.The ablation rate of lesions was calculated based on CEUS 24 h after MRgFUS.The focal blood flow score before,24 h after MRgFUS and the sum of the two,also the numerical rating scale(NRS)score before and 1,6 months after MRgFUS and the change rate were analyzed.The correlations of CEUS parameters with the efficacy of MRgFUS for treating adenomyoma were observed.Results No significant difference of lesion volume nor NPV on CEUS or MRI was found(both P＞0.05).The ablation rate of lesions 24 h after treatment was(58.11±24.92)%.The focal blood flow score before,24 h after MRgFUS and the sum of the two was 2.00(2.00,2.00),1.00(1.00,1.00)and 3.50(3.00,3.50),respectively,with significant difference between before and 24 h after MRgFUS(Z=-4.463,P＜0.001).NRS score was 5.00(4.00,6.00),3.00(2.00,4.00)and 2.00(1.00,3.00)before treatment,1 and 6 months after treatment,respectively,with significant differences at different time points(all P＜0.01).The change rate of NRS score 1 and 6 months after treatment was 35.42%(23.75%,50.00%)and 60.00%(50.00%,77.08%),respectively.The lesion blood flow score before and 24 h after MRgFUS and the sum of the two were all negatively correlated with ablation rate(rs=-0.552,-0.820,-0.745),while positively correlated with NRS scores 6 months after treatment(rs=0.513,0.552,0.496)but negatively correlated with the change rate of NRS scores 6 months after treatment(rs=-0.525,-0.479,-0.531).The ablation rate 24 h after treatment was negatively correlated with NRS scores(rs=-0.462)while positively correlated with the change rate of NRS scores 6 months after treatment(rs=0.500).Conclusion CEUS parameters before and after treatment were correlated with the therapeutic efficacy of MRgFUS for treating adenomyoma.

Objective:To evaluate the effect of activation of splenic plasmacytoid dendritic cells (pDCs) on myocardial ischemia-reperfusion (I/R) injury in mice.Methods:The experiment was performed in two parts. Animal experiment Thirty-six SPF healthy male C57BL/6J mice, aged 10 weeks, weighing 22-27 g, were assigned to 3 groups ( n=12 each) using a random number table method: sham operation group (Sham group), myocardial ischemia group (MI group) and myocardial I/R group (MI/R group). The myocardial ischemia was induced by occluding the left anterior descending coronary artery for 40 min in MI group, while the model of myocardial I/R was established by occlusion of the left anterior descending coronary artery for 40 min followed by 1-h reperfusion in MI/R group. Following successful preparation of the model, 3 animals from each group were randomly selected, and their hearts were removed for determination of myocardial infarct size through a combination of TTC and methylene blue double staining. Another 3 animals from each group were randomly selected, and their hearts were removed for examination of pathological changes of myocardial tissues using HE staining. Blood samples were collected from the abdominal aorta of 6 mice left in each group for determination of plasma interferon alpha (IFN-α) concentrations by enzyme-linked immunosorbent assay. Then the animals were sacrificed and hearts were harvested for collection of cardiac perfusate (CP). Cell experiment Twelve SPF healthy male C57BL/6J mice, aged 10 weeks, weighing 22-27 g, were selected and the splenic pDCs were isolated using anti-mPDCA-1 MicroBeads according to the manufacturer′s instructions (with a positivity rate of >85% for the isolated cells). The cells were divided into 4 groups: group pDCs stimulated by CP in Sham group (pDCs+ CP-Sham group), group pDCs stimulated by CP in MI group (pDCs+ CP-MI group), group pDCs stimulated by CP in MI/R group (pDCs+ CP-MI/R group) and pDCs stimulated by PBS group (pDCs+ PBS group). The CP in Sham, MI and MI/R groups and PBS were used to induce and culture pDCs for 8 h. Flow cytometry was employed to detect the expression of CD45 and co-stimulatory molecules CD80, CD86 and Major Histocompatibility Complex Ⅱ (MHC Ⅱ) on the surface of pDCs. The levels of IFN-α in the cell culture supernatant were determined using enzyme-linked immunosorbent assay. Results:Animal experiments Compared with Sham group and MI group, the percentage of myocardial infarct size was significantly increased, the concentrations of plasma IFN-α were increased ( P<0.05), and cardiomyocytes displayed evident vacuolar degeneration, severe myocardial fiber rupture, and infiltration of a substantial number of inflammatory cells in MI/R group. There was no significant difference in each parameter between Sham group and MI group ( P>0.05). Cell experiment Compared with pDCs+ CP-Sham group, the expression of CD80, CD86 and MHCⅡ was significantly up-regulated in pDCs+ CP-MI group ( P<0.05), and no significant change was found in the aforementioned parameters in pDCs+ CP-MI/R group ( P>0.05). The expression of aforementioned parameters was significantly up-regulated in pDCs+ CP-MI group as compared with pDCs+ CP-MI/R group ( P<0.05). Compared with pDCs+ CP-Sham group and pDCs+ CP-MI/R group, the concentrations of IFN-α in the cell culture supernatant were significantly increased in pDCs+ CP-MI group ( P<0.05). There was no statistically significant difference in the concentrations of IFN-α between pDCs+ CP-MI/R group and pDCs+ CP-Sham group ( P>0.05). Conclusions:The mechanism underlying myocardial I/R injury may be related to activation of splenic pDCs leading to the production of IFN-α following myocardial ischemia in mice.

Polycystic ovary syndrome(PCOS)is a heterogeneous disorder closely associated with reproductive endocrine dysfunction in the women.The etiology and pathogenesis of PCOS remain unclear.PCOS is the result of the combination of endocrine metabolic disorders,genetics,and environmental factors.Hyperandrogenemia(HA)and insulin resistance(IR)are the fundamental pathophysiological changes in the development of PCOS,and their interactions exacerbate the clinical manifestations of the PCOS patients.The family aggregation and twin study results confirm the genetic predisposition of PCOS;the genome-wide association study(GWAS)results confirm some risk loci and candidate genes of PCOS.The unhealthy lifestyle habits and environmental endocrine disruptors also play an important role in the progression of PCOS,and the gut microbita is involved in the pathogenesis of PCOS.This article provides a comprehensively retrospective analysis on the recent studies about PCOS,and reviews both internal factors and external factors related to the etiology and pathogenesis of PCOS.