[Effects of Triptolide on the Autophagy in Synovial, Spleen and Thymus of Rats with Adjuvant Arthritis].

OBJECTIVE: To determine the effect of triptolide (TP) on the expression of ATG /LC3-Ⅱ Beclin1 in synovial, spleen, and thymusof rats with adjuvant arthritis (AA). METHODS: Rats were divided for four groups: normal control (NC), model control (MC), leflunomide (LEF) treatment, and triptolide (TP)treatment, with 12 rats in each group.The AA model was established through Freund's complete adjuvant (0.1 mL each) injection into the right foot plantar skin to introduce inflammation and 10 days of tail root injection of 0.05 mL Freund's complete adjuvant for immunity strengthening. Drug administration started 13 days after induction of inflammation. Rats in the NC and MC groups were given normal saline (1 mL/100 g) once a day for 30 days, compared with 5 mg/kg of oral LEF for the rats in the LEF group and 50 µg/kg of oral TP for the rats in the TP group. Paw swelling (E), joint arthritis index(AI) and joint pathological changes of the rats were recorded. The serum expressions of cytokines B lymphocyte stimulating factor (BAFF), interleukin (IL)-1, tumor necrosis factor (TNF) alpha, IL-15,and IL-10 were detected by ELISA. The expressions of Atg5, Atg7, and Atg12 mRNA in synovial, spleen, and thymus of the rats were detected by RT-PCR.The expressions of LC3-Ⅱ and Beclin1 in synovial, spleen, and thymus of the rats were detected by Western blot assay. RESULTS: The AA model rats had lower serum BAFF, IL-1, TNF alpha, IL-15, and IL-10; lower Atg5and Atg12 mRNA in synovial; lower Atg5 mRNA, Atg7, and Atg12 mRNA in spleen; higher Atg12 mRNA in thymus; and lower LC3-Ⅱ and Beclin1 in synovial, spleen and thymus(P<0.05 or 0.01). TP treatment led to reduced paw swelling and arthritis index; declined Atg7 and Atg12 mRNA in synovial; declined Atg5, Atg7 mRNA and Atg12 mRNA in spleen; decreased Atg5 and Atg7mRNA in thymus; increased Atg12 mRNA in thymus; and increased LC3-Ⅱ and Beclin1 in synovial, spleen and thymus (P<0.05 or 0.01). Compared with rats treated with LEF, TP treated rats had lower TNF-α and BAFF and higher E and IL-15 (P<0.05 or 0.01); as well as decreased expressions of Atg7 mRNA (synovial) and Atg5, Atg7 mRNA (thymus), and increased expressions of Atg12 mRNA (thymus) and Atg5, Atg7, Atg12 mRNA (spleen). CONCLUSION: TP regulates autophagy in synovial, thymus and spleen of AA rats, and improves theirjointinflammatory response.

[Xinfeng Capsule Improved Blood Stasis State of Rheumatoid Arthritis Patients Based on Actl/NF-KB Signaling Pathway: Mechanism and Effects].

Objective To observe the mechanism of Xingfeng Capsule (XFC) for improving blood stasis state in rheumatoid arthritis (RA) patients based on Actl/NF-κB signaling pathway. Methods Totally 76 RA patients were equally assigned to two groups by random digit table, the XFC group (XFC, 3 pills each time, three times per day) and the Leflunomide group (LEF, 10 mg each time, once per day). All patients were intervened for 3 successive months. Clinical efficacy of symptoms of Chinese medicine (CM) was assessed. Serum levels of interleukin-10 (IL-10) , IL-17, IL-6, NF-κB activator 1 (Actl), p50, p65, platelet activating factor ( PAF) , platelet activating factor acetyl hydrolase ( PAF-AH) were detected using ELISA. Symptoms of blood stasis syndrome (BSS) were also assessed. mRNA expressions of Act1, p50, and p65 were detected using fluorescent quantitative PCR. Protein expressions of p50 and p65 were detected using Western blot. Correlation analyses were performed in RA patients' peripheral blood coagulation indicators, total score of BSS, and IL-1 0, IL-6, IL-17, Act1 , p50, p65 using Spearman. Results The total effective rate was 89. 5% (34t38) in the XFC group, with no statistical difference as compared with that of the LEF group [94. 7% (36)38), P >0. 05]. Compared with before treatment in the same group, serum levels of D-dimer (DD) , fibrinogen (FBG) , platelet (PLT) , PAF, IL-17, and IL-6 all decreased, mRNA expressions and serum levels of Act1, p50, and p65 were lowered, protein expres- sions of p50 and p65 decreased, scores for each symptoms in BSS all decreased, serum levels of PAF- AH and IL-10 increased in the two groups after treatment (P <0. 05, P <0. 01). Compared with the LEF group, serum levels of DD, FBG, PLT, IL-17, and IL-6 decreased, mRNA expressions and serum levels of Act1 and p65 were lowered, protein expression of p65 decreased, scores for joint prickling pain, tongue proper, subcutaneous ecchymosis, and BSS total score all decreased in the XFC group (P < 0. 05, P <0. 01). Peripheral blood DD was positively correlated with IL-17, IL-6, Act1, and and p65, but negatively correlated with IL-10 (P <0. 05, P <0. 01). FBG was positively correlated with IL-6 (P <0. 05). PLT was positively correlated with IL-17 (P <0. 05). BSS total score was positively correlated with IL-6, Act1, and p65, but negatively correlated with IL-10 (P <0. 05, P <0. 01). PAF was positively correlated with IL-17, IL-6, Act1 , and p65 (P <0. 05, P <0. 01), while PAF-AH was negatively correlated with p50 (P <0. 05). Conclusion The pathogenesis of BSS in RA patients and the effects of XFC on blood stasis state might be closely correlated to the Act1/NF-KB signaling pathway.

[Efficay Observation of Xinfeng Capsule for Treating 58 Cases of Sjogren's Syndrome].

Objective To observe the clinical effect of Xinfeng Capsule (XFC) in treating Sjögren's syndrome (SS) and its effect on coagulation parameters, peripheral blood cytokines, and NF- kappa B signaling pathway protein. Methods Totally 58 SS patients were assigned to the treatment group and the control group according to random digit table, 29 cases in each group. Patients in the treat- ment group took XFC, 3 pills each time, 3 times per day. Those in the control group took Hydroxychloro- quine ( HCQ) Sulfate, 0. 1 g per tablet, 2 tablets each time, twice per day. Three months consisted of one therapeutic course and one course for all. Another 20 healthy subjects were recruited as a normal control group. Coagulation parameters (APTT, PT, FIB,TT, D-D) were detected using automatic coagulation an- alyzer in the two groups before and after treatment as well as in the healthy control group. The expres- sions of peripheral blood cytokines (IL-1ß, TNF-α, IL-10) and NF-κB signaling pathway proteins (P65, P50, IκBα) were detected before and after treatment using ELISA. Erythrocyte sedimentation rate (ESR) was determined using Westergren method before and after treatment. High sensitivity C-reactive protein (hs-CRP) level was determined using automatic biochemical analyzer before and after treatment. Results Totally 44 SS patients had abnormal coagulation parameters, accounting for 75. 9% of the total. Com- pared with the healthy control group, the levels of D-D, FIB, IL-1 , TNF-α,P50, P65, IκBα, hs-CRP, and ESR increased (P <0.05, P <0. 01), and the IL-10 level decreased in SS patient groups (P <0.01). Spearman correlation analysis showed that coagulation parameters were significantly correlated with cy- tokines, NF-κB signal transduction pathway, and inflammation indices (P <0. 01 , P <0. 05). After drug in- tervention blood coagulation parameters and laboratory indices were partially improved in the two groups. The effective rate and the total effective rate were 59% and 86% in the treatment group, obviously higher than those of the control group with statistical difference (38% and 72%; P <0. 05). Besides, the treat- ment group was superior to the control group in reducing the levels of FIB, D-D, P50, P65, ESR, and hs- CRP, down-regulating levels of TNF-α and IL-1 ß, as well as up-regulating the expression of IL-10 (P < 0. 05, P <0. 01). Conclusions There is a hypercoagulable state in SS patients, which is related to abnor- mal activation of cytokines/NF-kappa B signaling pathway. XFC could effectively improve the hypercoagulative state of SS patients. Its mechanism might be related to inhibiting cytokines/NF-κB signaling pathway.

[Effect of Xinfeng Capsule in Improving Blood Stasis State of OA Patients].

Objective To observe the relationship between blood stasis state and activation of nuclear factor-κ-gene binding (NF-κB) signaling pathway/miRNA-146 in osteoarthritis (OA) patients and the effect of Xinfeng Capsule (XFC) on them. Methods Totally 70 OA patients were assigned to the treatment group and the control group according to random number table, 35 in each group. Patients in the treatment group took XFC, 3 pills each time, 3 times per day, while those in the control group took Glucosamine, 1 pill each time, 3 times per day. The therapeutic course for all was 3 months. Serum con- tents of p50, p65, inhibitor of nuclear factor κB O (IκBα) , nuclear factor kappa B activator 1 (Act1) , TGF-ß-activated kinase 1 (TAK1) , IL-1, IL-17, IL-10, and thromboxane A2(TXA2) , prostacycline (PGI2) were detected by ELISA. mRNA levels of Act1 , p65, p50, and TAK1 were detected using fluorescent quantitative PCR. The protein levels of p50 and p65 were detected using Western blot. The level of miRNA- 146 was detected using in one-step PCR. Results (1) Compared with pre-treatment in the same group, the levels of blood stasis score, platelets (PLT) , D-dimer (D-D) , TXA2, IL-1, IL-17, high-sensitivity C- reactive protein (hs-CRP), and erythrocyte sedimentation rate (ESR) all decreased; mRNA levels of p50, p65, Act1, and TAK1 were lowered; protein expressions of p50 and p65 decreased; serum levels of miRNA-146 decreased; activated partial thromboplastin time (APTT) , prothrombin time ( PT) , prosta- glandin 2 (PGI2), IL-10 increased in the two groups after treatment with statistical difference (P <0. 05, P <0. 01). Compared with the control group, the levels of blood stasis score, PLT, FIB,TXA2, IL-17, hs- CRP, and ESR were lowered; mRNA expressions of p65 and TAK1 were lowered; protein expressions of p50 decreased; levels of PT and PGI2 increased in the treatment group after treatment (P <0. 05, P < 0.01). Conclusion XFC could regulate the immunity and restore the equilibrium of cytokine network, and protect vascular endothelial cells possibly by up-regulating miRNA-146 expression and inhibiting acti- vation of NF-κB signaling pathway, thus improving blood stasis state of OA.

Proteomics analysis of lung tissue reveals protein makers for the lung injury of adjuvant arthritis rats.

Lung injury is one of the common extra­articular lesions in rheumatoid arthritis (RA). Due to its insidious onset and no obvious clinical symptoms, it can be easily dismissed in the early stage of diagnosis, which is one of the reasons that leads to a decline of the quality of life and subsequent death of patients with RA. However, its pathogenesis is still unclear and there is a lack of effective therapeutic targets. In the present study, tandem mass tag­labeled proteomics was used to research the lung tissue proteins in RA model (adjuvant arthritis, AA) rats that had secondary lung injury. The aim of the present study was to identify the differentially expressed proteins related to RA­lung injury, determine their potential role in the pathogenesis of RA­lung injury and provide potential targets for clinical treatment. Lung tissue samples were collected from AA­lung injury and normal rats. The differentially expressed proteins (DEPs) were identified by tandem mass spectrometry. Bioinformatic analysis was used to assess the biological processes and signaling pathways associated with these DEPs. A total of 310 DEPs were found, of which 244 were upregulated and 66 were downregulated. KEGG anlysis showed that 'fatty acid degradation', 'fatty acid metabolism', 'fatty acid elongation', 'complement and coagulation cascades', 'peroxisome proliferator­activated receptor signaling pathway' and 'hypoxia­inducible factor signaling pathway' were significantly upregulated in the lung tissues of AA­lung injury. Immunofluorescence staining confirmed the increased expression of clusterin, serine protease inhibitors and complement 1qc in lung tissue of rats with AA lung injury. In the present study, the results revealed the significance of certain DEPs (for example, C9, C1qc and Clu) in the occurrence and development of RA­lung injury and provided support through experiments to identify potential biomarkers for the early diagnosis and prevention of RA­lung injury.