Chemiluminescence method was used to measure: (1) active oxygen species generation induced by respiratory burst of polymor-phonuclear leucocytes (PMNs) from human blood stimulated with phorbol myristate acetate (PMA); (2) superoxide (O2) induced by xan-thine-xanthine oxidase system; (3) hydroxyl radicals ( ? OH ) generated by Vit C- Cu2+- zy-mosan; (4) the release of hydrogen peroxide (H2O2). Effects of polydatin IV on these active oxygen species were observed. The resultsshowed early stage of respiratory burst of PMNs was inhibited,but the later stage was delayed by polydatin IV, (2), (3) adn (4) were scavenged by polydatin IV and their median inhibitory concentrations (IC50?mol ? L-1) were 14.6,29.6 and 13.0 respectively. The results suggested that polydatin IV was a scavenger.

Periodontal disease (PD) is an infection-driven chronic inflammatory disease characterized by the inflammation of tooth-supporting tissues and the destruction of the associated alveolar bone. The immune response of the host to periodontal pathogens infection determines the course and progress of the disease. The effects of secreting cytokines interferon-gamma (IFN-γ) and interleukin-17 (IL-17) of T helper 1 cells (Th1) and T helper 17 cells (Th17) on the development of periodontitis has attracted much attention. IFN-γ is a potential immune-modulatory cytokine and can mediate cellular immune responses by activating various immune cells of the host such as macrophages. As one of the most potential bone physiological regulation mediators, IL-17 is closely related with alveolar bone resorption in periodontitis. This review elaborated the relationship between IFN-γ and IL-17 in the progress of periodontitis, providing new explanations into the development of periodontitis and alveolar bone destruction caused by the host immune response.

To investigate the effects of interleukin (IL)-17-mediated autophagy on the TNF receptor associated factor (TRAF6)/extracellular signal-regulated kinase (ERK)/p38 pathway and osteoclast differentiation. Mouse bone marrow-derived macrophages (BMM) were cultured with a medium containing 30 ng/mL macrophage colony stimulating factor and 50 ng/mL receptor activator of nuclear factor-kappa B ligard (RANKL), and IL-17 (0.01, 0.1, 1.0, 10 ng/mL) was added for intervention (IL-17 group). Tartrate-resistant acid phosphatase (TRAP) staining was used to observe TRAP positive multinucleated cells; phalloidin fluorescent staining was used to detect actin ring circumference; toluidine blue staining was used to analyze bone resorption lacuna formation. To further examine the mechanism of the effect of IL-17-mediated autophagy on the differentiation of osteoclasts, the control group used RANKL medium to culture mouse macrophage RAW264.7 cells, while the IL-17 group was treated with IL-17 (0.01, 0.1, 1.0, /mL). Western blot was used to detect the expression of autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) and osteoclast-related proteins c-fos and nuclear factor of activated T cell 1 (NFATc1) after treatment with different concentrations of IL-17. The expression of LC3, NFATc1, TRAF6/ERK/p38 signaling pathway related proteins were detected in IL-17 and autophagy inhibitor 3-MA group. The number of TRAP positive multinucleated cells, the circumference of the actin ring and the area of bone resorption lacuna in IL-17 group treated with IL-17 (0.01, 0.1, were significantly higher than those in the control group. In IL-17 treated RAW264.7 cells, the expression of c-fos, NFATc1, Beclin-1, LC3, TRAF6, p-ERK, and p-p38 was all significantly up-regulated (all 0.05). After treatment with the autophagy inhibitor 3-MA, the expression levels of LC3, NFATc1, TRAF6, p-ERK, and p-p38 all decreased significantly (all 0.05). IL-17 can promote the expression of autophagy proteins and enhance the differentiation ability of osteoclast precursor cells, and the TRAF6/ERK/p38 signaling pathway may be involved in this process.
Animals ; Autophagy ; Bone Resorption ; Cell Differentiation ; Extracellular Signal-Regulated MAP Kinases ; Interleukin-17 ; Mice ; NFATC Transcription Factors/metabolism* ; Osteoclasts/metabolism* ; RANK Ligand/metabolism* ; TNF Receptor-Associated Factor 6

Objectvie: To investigate the effects of secreting cytokines interferon-gamma (IFN-γ) and interleukin-17 (IL-17) of T helper 1 cells (Th1) and T helper 17 cells (Th17) on the peripheral blood and alveolar bone destruction, so as to provide a new explanation for cellular immunity-mediated alveolar bone destruction. Methods: Eighteen eight-week-old male Sprague-Dawley rats were divided, randomly and equally, into 3 groups: lipopolysaccharide (LPS) group, ligation group and normal control group. In the LPS group, Escherichia coli LPS was injected into the alveolar mucosa on the buccalmedian site of the left upper first molar, while the right upper first molar was injected with equal volume of physiological saline as self-controls. The injections were performed every other day for four times totally. In the ligation group, the left upper first molars were ligatured with 0.2 mm orthodontic cords, while the right upper first molars were left untreated as self-controls, and supplemented with high-sugar diet to promote the periodontitis status. The rats in normal control group were fed normally. The concentrations of IFN-γ and IL-17 in peripheral blood were measured using enzyme linked immunosorbent assay (ELISA) method at the fourth week after the start of injection and at the eighth week after ligation. The histological of periodontal tissues were observed after hematoxylin-eosin (HE) staining and osteoclast count was performed under light microscope. The histological of osteoclasts were observed after tartrate-resistant acid phosphatase (TRAP) staining. Expression of IFN-γ and IL-17 were detected by immunohistochemical assay. Results: The concentrations of IFN-γ in peripheral blood of LPS group [(185.0±50.7) ng/L] and ligation group [(202.9±60.4) ng/L] were significantly higher than that of normal control group [(106.3±17.2) ng/L](P<0.05). Meanwhile, histological examination showed inflammatory cells infiltration in the gingival epithelium, the height reduction of alveolar bone accompanied with absorption lacuna. There were significantly higher HE and TRAP stained osteoclasts in LPS group (9.50±1.05) and ligation group (10.83±1.17) than that in controlgroup (0.33±0.52)(P<0.05). Moreover, the expressions of IL-17 in alveolar bone absorption area of LPS group and ligation group were significantly stronger than that in control group (P<0.05). Conclusions The rat models of experimental periodontitis and alveolar bone resorption could be successfully established by means of ligationand LPS injection, respectively. The periodontal inflammatory responses were related to secreting cytokines IFN-γ and IL-17 of Th1 and Th17 cells, while Th17 cells might exert a positive effect on alveolar bone destruction.

To analyze the global burden of periodontal disease and its relation with socioeconomic development. Data of global disability-adjusted life year (DALY) due to periodontal disease and human development index (HDI) from 1990 to 2019 were obtained from Global Health Data Exchange (GHDx) and human development reports. The trend of the global burden of periodontal disease from 1990 to 2019 was described. The correlation between age-standardized DALY rates and HDI were examined in 2019, and between-country periodontal disease burden inequality from 1990 to 2019 was measured using health-related Gini coefficients and concentration indexes. From 1990 to 2019, the global DALY rate due to periodontal disease increased from 78.63 to 85.48, and the epidemiological burden did not increase significantly. Statistical differences were found across different HDI categories for age-standardized DALY rates of periodontal disease ( 44.315, <0.01) in 2019. Linear regression analysis also revealed a negative correlation between age-standardized DALY rate of periodontal disease and HDI ( = -0.417, <0.01) . Gini coefficients decreased from 0.361 to 0.281 and concentration indexes fell from 0.0339 to -0.0538 between 1990 and 2019. The global burden of periodontal disease did not increase between 1990 and 2019, though the socioeconomic-associated inequality still existed. The burden of periodontal disease was more concentrated in less developed countries, and the socioeconomic-associated inequality has increased since 2000.
Disability-Adjusted Life Years ; Global Health ; Humans ; Periodontal Diseases/epidemiology* ; Quality-Adjusted Life Years ; Socioeconomic Factors

Objective     To search for the key microRNAs (miRNAs) involved in myocardial fibrosis in hypertrophic cardiomyopathy, and to further explore the mechanisms involved in the regulation of myocardial fibrosis. Methods    Forty-two patients with hypertrophic cardiomyopathy diagnosed and treated surgically in West China Hospital of Sichuan University from January 2014 to June 2018 were selected, including 29 males and 13 females, with a median age of 46 (15-69) years. In the myocardial tissue of patients with hypertrophic cardiomyopathy with different degrees of fibrosis, miRNAs with significantly different expression were screened and further verified at the cellular level. By regulating the expression of the target miRNAs, the expressions of fibrosis-related proteins and target genes were detected respectively. Finally, the target-binding relationship was verified by dual-luciferase reporter gene detection. Results    miR-484 was up-regulated in severely fibrotic myocardial tissue and activated cardiac fibroblasts. After cardiac fibroblasts were activated by TGF-β1, the expression of miR-484 was significantly up-regulated, the expression of fibrosis-related proteins (CollagenⅠ, α-SMA) increased, and the expression of the target gene HIPK1 decreased (P<0.05). After inhibiting the expression of miR-484 by transfection of miR-484 antagomir, the expression of fibrosis-related proteins decreased, while expression of HIPK1 was up-regulated (P<0.05). The detection of dual luciferase reporter gene showed that the luciferase activity of the transfected WT-miRNA-484 mimics group was lower than that of the control group (P<0.05). Conclusion    miR-484 is a pro-fibrotic miRNA that participates in the process of myocardial fibrosis by negatively regulating the expression of HIPK1. It can be used as a regulatory target to provide a therapeutic strategy for myocardial fibrosis.

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.
Gene Editing ; Aspergillus niger/genetics* ; CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg^-1·d^-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg^-1·d^-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.