TLR9 regulates the autophagy-lysosome pathway to promote dendritic cell maturation and activation by activating the TRAF6-cGAS-STING pathway.

Dysregulated maturation and activation of dendritic cells (DCs) play a significant role in the progression of systemic lupus erythematosus (SLE). The autophagy-lysosome pathway has been identified as a potential mechanism to inhibit DC activation and maturation, but its precise workings remain unclear. We investigated the role and regulatory mechanism of TLR9 in modulating the autophagy-lysosome pathway and DCs activation. The mRNA and protein expressions were assessed using qRT-PCR and/or western blot. NZBW/F1 mice was used to construct a lupus nephritis (LN) model in vivo. Cell apoptosis was analyzed by TUNEL staining. Flow cytometry was adopted to analyze DCs surface markers. Lyso-tracker red staining was employed to analyze lysosome acidification. Levels of anti-dsDNA, cytokines, C3, C4, urine protein and urine creatinine were examined by ELISA. The results showed that TLR9 was markedly increased in SLE patients, and its expression was positively correlated with SLEDAI scores and dsDNA level. Conversely, TLR9 expression showed a negative correlation with C3 and C4 levels. Loss-of function experiments demonstrated that TLR9 depletion exerted a substantial inhibition of renal injury, inflammation, and DCs numbers. Additionally, upregulation of TLR9 promoted DCs maturation and activation through activation of autophagy and lysosome acidification. Further investigation revealed that TLR9 targeted TRAF6 to activate the cGAS-STING pathway. Rescue experiments revealed that inactivation of the cGAS/STING signaling pathway could reverse the promoting effects of TLR9 upregulation on DCs maturation, activation, and autophagy-lysosome pathway. Overall, our findings suggested that TLR9 activated the autophagy-lysosome pathway to promote DCs maturation and activation by activating TRAF6-cGAS-STING pathway, thereby promoting SLE progression.

LncRNA GAS5 alleviates rheumatoid arthritis through regulating miR-222-3p/Sirt1 signalling axis.

INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease that affects millions of people. Fibroblast-like synoviocytes (FLSs) located in rheumatoid panni play a pivotal role in the formation of RA. The long noncoding RNA (lncRNA) GAS5 is reportedly downregulated in rheumatoid arthritis. However, its detailed mechanism in RA remains to be explored. This study investigated the roles and related mechanisms of GAS5 in RA. METHODS: The expression levels of GAS5, miR-222-3p, and sirtuin 1 (Sirt1) were evaluated by quantitative PCR (qPCR). Cell proliferation was analyzed by CCK-8 and BrdU assays. Cell apoptosis was assessed by flow cytometry and western blotting. Enzyme-linked immunosorbent assay (ELISA) was utilized to evaluate the levels of TNF-α, IL-1ß, and IL-6. The interaction between GAS5 or Sirt1 and miR-222-3p was predicted by starBase and validated by dual-luciferase reporter assay. RESULTS: GAS5 expression was found to be downregulated in the serum samples of RA patients and in RA-FLSs. GAS5 overexpression or the inhibition of miR-222-3p impeded the activity of RA-FLSs by repressing their proliferation and inflammation and by promoting apoptosis. Mechanistically, GAS5 indirectly regulates Sirt1 expression by binding miR-222-3p. Further experiments confirmed that Sirt1 overexpression restored the anti-RA activity of GAS5 under miR-222-3p mimic. CONCLUSIONS: The miR-222-3p/Sirt1 axis was found to be critical for the function of GAS5 in regulating the proliferation, inflammation, and apoptosis of RA-FLSs. These data indicate GAS5 activation as a potential therapeutic strategy for RA progression.

[Changes of the activity and expression of gamma-glutamylcysteine synthetase in patients with chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the oxidative/antioxidative status in patients with chronic obstructive pulmonary disease (COPD), and to study the expression and location of gamma-glutamylcysteine synthetase (gamma-GCS) in lung tissues. METHODS: (1) Serum samples of 13 patients with COPD in exacerbation and 9 healthy non-smokers were collected for measurements of the level of reduced glutathione (GSH), reactive oxygen species (ROS), total antioxidative capacity (T-AOC) and gamma-GCS activity. (2) Lung tissues from 22 patients (12 patients with COPD and 10 patients as control) undergoing resection for lung tumor were collected for study of the expression and location of gamma-GCS and gamma-GCS mRNA by immunohistochemistry and in situ hybridization, respectively. RESULTS: (1) Significantly decreased level of GSH [(23.87 +/- 3.86) mg/ml], increased level of ROS [(2 463 +/- 199) U/ml], decreased T-AOC [(5.34 +/- 0.22) U/ml] and enhanced activity of gamma-GCS [(19.22 +/- 3.36) U/ml] were shown in the serum of patients with an acute exacerbation of COPD, as compared to the control group [(36.87 +/- 6.34) mg/ml, (1 023 +/- 112) U/ml, (11.36 +/- 1.07) U/ml and (12.37 +/- 2.96) U/ml, respectively, all P < 0.05]. (2) There was no relationship between the level of GSH, ROS, T-AOC, gamma-GCS activity in serum and FEV1%, FEV1/FVC, PaO2, PaCO2 of patients with COPD in exacerbation (P > 0.05). (3) In situ hybridization showed that the expressions of gamma-GCS mRNA in alveoli, bronchi and inflammatory cells (A value was 0.29 +/- 0.05, 0.31 +/- 0.05 and 0.28 +/- 0.06, respectively) from the COPD group were stronger than those from the control group (0.14 +/- 0.03, 0.17 +/- 0.04 and 0.20 +/- 0.05, respectively) by semi-quantitative analysis (all P < 0.05). (4) By immunohistochemistry, the expressions of gamma-GCS was significantly higher in alveoli (0.20 +/- 0.04), bronchi (0.18 +/- 0.02) and inflammatory cells (0.25 +/- 0.06) in the COPD group as compared to the control group (0.12 +/- 0.04, 0.10 +/- 0.03 and 0.14 +/- 0.04, respectively, all P < 0.05). (5) Negative correlations were shown between gamma-GCS and FEV1%, FEV1/FVC (r = - 0.501 and - 0.542, respectively, P < 0.05), while the level of gamma-GCS expression had no correlation with FEV1% and FEV1/FVC (r = - 0.221 and - 0.148, respectively, P > 0.05), and a positive relationship was observed between gamma-GCS and gamma-GCS mRNA (r = 0.732, P < 0.05). CONCLUSIONS: Systemic oxidative/antioxidative imbalance occurs in patients with acute exacerbation of COPD, and the total antioxidative capacity decrease may not correlate significantly to the obstruction of airways, in spite of the high level of gamma-GCS activity in serum and gamma-GCS expression in the lungs.

[Fosinopril up-regulates and ameliorates the Ang II induced down-expression of klotho gene in NRK-52E].

OBJECTIVE: To investigate the effect of fosinopril (Fos) on regulating klotho gene expression and elucidate the mechanism of Fos regulating the Angiotensin II (AngII) -induced down-expression of klotho gene. METHODS: Culture cells, NRK-52E, were incubated with media either AngII or Fos or both of all. Experimental groups incubated with Fos (10(-5) mol/L) were divided according to variant points of time for 0 (control), 3, 6, 12, 24 h. Different concentration of Fos was selected to incubated with culture cells for 0 (control), 10(-9) 10(-8), 10(-7), 10(-6), 10(-5) mol/L at the optimal time point (24 h). Five groups, which were A: control; B: AngII (10(-7) mol/L); C: Fos(10(-5) mol/L); D: AngII (10(-7) mol/L) + Fos(10(-5) mol/L) and E: Cells pretreated with Fos(10(-5) mol/L)12 h incubated with AngII (10(-7) mol/L) were divided to observe the effect of Fos on expression of klotho induced by AngII. RT-PCR and immunohistochemistry (IHC) were applied to evaluate the klotho mRNA and protein expression, respectively. RESULTS: Fos up-regulated klotho mRNA in time-dependent manner, and independent of dose-dependent manner; AngII obviously decreased the levels of kloltho mRNA and protein expression in NRK-52E as compared to the control (P < 0.05), the down-regulating effect was reversed by incubating both with AngII and Fos (P < 0.05), and Fos could inhibit the down-regulated expression of klotho gene induced by Ang II in NRK-52E. CONCLUSION: Fosinopril up-regulates klotho mRNA in time-dependent manner, and inhibits the down-regulated expression of klotho gene induced by Ang II.

[Therapeutic effect of Tripterygium wilfordii multiglycosides on proteinuria in kidney transplant recipients].

OBJECTIVE: To study the effect of multiglycosides of Tripterygium wilfordii (MTW) for treatment of proteinuria in kidney transplant recipients. METHOD: Forty-five kidney transplant recipients with proternuria were randomized into 3 groups (n=15) and received full daily dose (1 mg/kg) MTW, half dose (0.5 mg/kg) MTW or no MTW (control) in addition to immunosuppressant therapy. The 24-hour urinary protein (24 h Upro), blood urea nitrogen (BUN), serum creatinine (Scr), dose of ciclosporin and the adverse effects of MTW were recorded. RESULTS: MTW at both the full dose and half dose significantly reduced the 24 h Upro as compared to exclusive immunosuppressant therapy (P<0.05). The therapeutic dose of ciclosporin in patients with full and half dose of MTW was significantly lower than that in the control group (P<0.05), and the patients receiving full dose MTW showed greater adverse effects than those having half dose MTW (P<0.05). CONCLUSIONS: MTW can significantly ameliorate proteinuria, reduce the therapeutic dose of ciclosporin and protect the renal function in kidney transplant recipients. While producing similar therapeutic effect to routine full dose, long-term use of half dose MTW may reduce the adverse effect associated with MTW.

[Location and expression of NF-kappaB in lung of rats with chronic obstructive pulmonary disease].

AIM: To elucidate the location and effects of transcription factor-nuclear factor-kappaB (NF-kappaB) in lung tissues of rats with chronic obstructive pulmonary disease (COPD). METHODS: Fourteen male Wistar rats were randomly divided into COPD model and control groups equally. The COPD model was established by intratracheal instillation of lipopolysaccharide twice and exposure to cigarette smoke daily. We detected the NF-kappaB p65 protein in lung by immunohistochemical method, and the expression of NF-kappaB p65 mRNA in lung by in situ hybridization. RESULTS: Immunohistochemistry, the expression of NF-kappaB p65 protein in alveolar, bronchiolar epithelium and arteriolar endothelium was significantly higher in the COPD group (0.426 +/- 0.007, 0.434 +/- 0.012 and 0.313 +/- 0.007, respectively) than those of the control group (0.115 +/- 0.006, 0.116 +/- 0.005 and 0.095 +/- 0.007, respectively, all P < 0.01). In situ hybridization showed that the expressions of NF-kappaB p65 mRNA in alveolar epithelium (0.203 +/- 0.008), bronchiolar and arteriolar smooth muscle cell (0.208 + 0. 010 and 0.206 + 0.007) of rats in the COPD group were stronger than those in the control group (0.100 +/- 0.006, 0.102 +/- 0.002 and 0.103 +/- 0.003 respectively) by semiquantitative analysis (all P < 0.01). CONCLUSION: The expression and nuclear translocation of NF-kappaB may be the basis event of gene expression of many cytokines and inflammatory mediators, which may positively regulate gene expression of many cytokines and inflammatory mediators in various cell lines.