CCN1 Promotes Inflammation by Inducing IL-6 Production via α6ß1/PI3K/Akt/NF-κB Pathway in Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease with unknown etiology. CCN1, an extracellular matrix-associated protein, is associated with carcinoma, inflammation, liver fibrosis, and even autoimmune diseases. However, the role that CCN1 plays in AIH has remained undetermined. In this study, expression of CCN1 in liver was detected by real-time PCR, western blot and immunohistochemistry (IHC). CCN1 level in serum was detected by ELISA. Diagnostic value of CCN1 was determined by receiver operating characteristic (ROC) curve analysis. CCN1 conditional knockout (CCN1 fl/fl Cre+) mice were generated by mating CCN1 fl/fl C57BL/6J and CAG-Cre-ERT C57BL/6J mice. Autoimmune hepatitis mice model was induced by concanavalin A (ConA). IKKα/ß, IκBα, NF-κB p65 and Akt phosphorylation were determined by western blot. NF-κB p65 nuclear translocation was examined by immunofluorescence. Here, we found that CCN1 was over-expressed in hepatocytes of AIH patients. CCN1 level also increased in serum of AIH patients compared to healthy controls (HC). ROC curve analysis results showed that serum CCN1 was able to distinguish AIH patients from HD. In ConA induced hepatitis mice model, CCN1 conditional knockout (CCN1 fl/fl Cre+) attenuated inflammation by reducing ALT/AST level and IL-6 expression. In vitro, CCN1 treatment dramatically induced IL-6 production in LO2 cells. Moreover, the production of IL-6 was attenuated by CCN1 knockdown. Furthermore, we showed that CCN1 could activate IL-6 production via the PI3K/Akt/NF-κB signaling pathway by binding to α6ß1 receptor. In summary, our results reveal a novel role of CCN1 in promoting inflammation by upregulation of IL-6 production in AIH. Our study also suggests that targeting of CCN1 may represent a novel strategy in AIH treatment.

iTRAQ-based proteomic analysis of differentially expressed proteins in sera of seronegative and seropositive rheumatoid arthritis patients.

OBJECTIVE: The diagnosis of seronegative rheumatoid arthritis (SNRA) is often difficult due to the unavailability of reliable laboratory markers. The aim of this study was to identify differentially expressed proteins in sera of SNRA, seropositive RA (SPRA), and healthy donors (HD). METHODS: A total of 32 seropositive RA patients, 32 SNRA patients, and 35 HD were enrolled in our study. Differentially expressed proteins between 3 groups were identified via isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis, and an ELISA test was used for the validation test. Correlation analysis was conducted by GraphPad Prism. RESULTS: Using iTRAQ quantitative proteomics, we identified 14 proteins were significantly different between SPRA and SNRA, including 4 upregulated proteins and 10 downregulated proteins. Four differentially expressed proteins were validated by ELISA test, and the results showed that SAA1 protein was significantly higher in SPRA and SNRA patients compared with HD, and PSME1 was elevated in SPRA patients. What's more, SAA1 was increased in the anti-CCP or RF high-level group in RA patients, and PSME1 was increased in the RF high-level group. Alternatively, SAA1 was positively correlated with inflammation indicators in RA patients, while PSME1 showed no correlation with inflammation indicators. CONCLUSIONS: iTRAQ proteomic approaches revealed variations in serum protein composition among SPRA patients, SNRA patients, and HD and provided new idea for advanced diagnostic methods and precision treatment of RA.

YY1 regulation by miR-124-3p promotes Th17 cell pathogenicity through interaction with T-bet in rheumatoid arthritis.

Th17 cells are involved in rheumatoid arthritis (RA) pathogenesis. Our previous studies have revealed that transcription factor Yin Yang 1 (YY1) plays an important role in the pathogenic mechanisms of RA. However, whether YY1 has any role in Th17 cell pathogenicity and what molecular regulatory mechanism is involved remain poorly understood. Here, we found the proportion of pathogenic Th17 (pTh17) cells was significantly higher in RA than in control individuals and showed a potential relationship with YY1 expression. In addition, we also observed YY1 expression was increased in pTh17, and the pTh17 differentiation was hampered by YY1 knockdown. Consistently, knockdown of YY1 decreased the proportion of pTh17 cells and attenuated joint inflammation in collagen-induced arthritis mice. Mechanistically, YY1 could regulate the pathogenicity of Th17 cells through binding to the promoter region of transcription factor T-bet and interacting with T-bet protein. This function of YY1 for promoting pTh17 differentiation was specific to Th17 cells and not to Th1 cells. Moreover, we found miR-124-3p negatively correlated with YY1 in RA patients, and it could bind to 3'-UTR regions of YY1 to inhibit the posttranscriptional translation of YY1. Altogether, these findings indicate YY1 regulation by miR-124-3p could specifically promote Th17 cell pathogenicity in part through interaction with T-bet, and these findings present promising therapeutic targets in RA.

An explanation for fluctuations of icariin content in Epimedium production process.

Epimedium has beneficial effects in nourishing and building up the body and is widely used in practical production of Epimedium preparations. As one of the major active compounds in Epimedium preparations, icariin is be used as a quality control index of industrial manufacture. However, content of icariin was observed to increase to uncertain extent in pharmaceutical production, which might bring difficulties in quality control. The content fluctuation mainly occurred in high-temperature extraction process. The aim of this study is to investigate what happen to flavonol-glycosides in Epimedium under heating treatment. Ultra-Performance Liquid Chromatography-Linear Ion Trap Mass Spectrometer was applied to profile the transformation rule of flavonol-glycosides in Epimedium and search for an explanation for the increase in icariin content under heating treatment. 56 compounds were found to have significantly changed and their structures were identified, among which 15 flavonol-glycosides were proposed to play a role in icariin content variation. Further studies were conducted based on 8 flavonol-glycosides standard substances to obtain more credible data. Finally, Baohuoside II, 2"-o-rhamnosylicariside II, Epimedin A1, Epimedin A, Epimedin B, Epimedin C, Baohuoside I and Anhydroicaritin were found to transform into icariin during the heating process. This study provides an evidence for the quality control study of Epimedium preparation, as well as reference for chemical researches in natural pharmacy.

Alteration of the gut microbiota in tumor necrosis factor-α antagonist-treated collagen-induced arthritis mice.

AIM: Gut microbiota play an important role in rheumatoid arthritis (RA). Biological therapies targeting tumor necrosis factor-α (TNF-α) have been used for treatment in RA patients. However, whether TNF-α antagonist has some influence on gut microbiota is still unknown. This study aims to investigate the distribution of gut microbiota in collagen-induced arthritis (CIA) mice treated with the TNF-α antagonist etanercept. METHODS: Collagen-induced arthritis mice were induced by type II collagen. Cytokine expression was detected by real-time polymerase chain reaction. 16S ribosomal RNA sequencing was performed to characterize the gut microbiota in CIA mice treated with vehicle or etanercept. Sequencing reads were processed by Microbial Ecology software program. RESULTS: Compared with vehicle-treated mice, we showed that CIA mice treated with etanercept led to attenuation of inflammation and reduced expression of TNF-α, interferon (IFN)-γ, interleukin (IL)-6 and IL-21. Meanwhile, results showed operational taxonomic units, richness estimators and the diversity indices of gut microbiota in etanercept-treated mice were lower than that in vehicle-treated mice. Moreover, bacterial abundance analyses showed that genus Escherichia/Shigella was more abundant in etanercept-treated mice, and Lactobacillus, Clostridium XlVa, Tannerella were less abundant. The altered bacterial genus was correlated with TNF-α, IFN-γ, IL-6, IL-21 and IL-10. CONCLUSION: Our results revealed that TNF-α antagonist treatment can reduce the abundance and diversity of gut microbiota in CIA mice. Targeted gut microbiota may be a new therapeutic strategy for the treatment of RA.

Correlation between albumin to fibrinogen ratio, C-reactive protein to albumin ratio and Th17 cells in patients with rheumatoid arthritis.

BACKGROUND: The albumin to fibrinogen ratio (AFR) and the C-reactive protein to albumin ratio (CAR) have been served as inflammatory markers. However, their roles in RA remain unclear. We investigated the association of AFR/CAR with the concentration of autoantibodies and Th17 cells in RA. METHODS: A total of 196 RA patients, 200 patients with systemic lupus erythematosus (SLE), and 200 healthy donors (HD) who were admitted to the First Affiliated Hospital of Fujian Medical University were enrolled. The results of FIB, ALB, CRP, anti-cyclic citrullinated peptide antibodies (anti-CCP), rheumatoid factor (RF) and erythrocyte sedimentation rate (ESR) from RA patients and SLE patients were retrospectively analyzed. The percentage of Th17 cells in peripheral blood of RA patients was detected by flow cytometry, and the relative expression of TNF-α, IL-6 and IL-17A was detected by RT-qPCR. Correlation analysis of AFR/CAR and Th17 cells, CRP, ESR, TNF-α, IL-6 and IL-17A in RA was conducted. RESULTS: Compared with SLE patients and healthy donors (HD), AFR concentration was significantly lower (P < 0.01) in RA patients, while CAR concentration was significantly increased (P < 0.01) in RA patients. AFR showed negative correlation with CRP (r = -0.7103), ESR (r = -0.6542), RF (-0.2219), Th17 cells (r = -0.5952) and IL-17A (r = -0.4681). CAR was positively correlated with CRP (r = 0.9899), ESR (r = 0.605), RF (0.1867), Th17 cells (r = 0.6818), TNF-α (r = 0.3388), and IL-17A (r = 0.2046). CONCLUSIONS: The concentration of AFR in RA patients was reduced, while CAR concentration was increased. AFR and CAR are associated with CRP, ESR, RF, and Th17 cell ratios in RA patients, which can be used as potential indicators for determining RA inflammation.

Establishment of Coamplification at Lower Denaturation Temperature PCR/Fluorescence Melting Curve Analysis for Quantitative Detection of Hepatitis B Virus DNA, Genotype, and Reverse Transcriptase Mutation and Its Application in Diagnosis of Chronic Hepatitis B.

Dynamic and real-time hepatitis B virus (HBV) DNA, genotype, and reverse transcriptase mutation analysis plays an important role in diagnosing and monitoring chronic hepatitis B (CHB) and in assessing the therapeutic response. We established a highly sensitive coamplification at lower denaturation temperature PCR (COLD-PCR) coupled with probe-based fluorescence melting curve analysis (FMCA) for precision diagnosis of CHB patients. The imprecision with %CV and detection limit of HBV DNA detected by COLD-PCR/FMCA were 2.58% to 4.42% and 500 IU/mL, respectively. For mutation, the imprecision and detection limit were 3.35% to 6.49% and 1%, respectively. Compared with Sanger sequencing, the coincidence rates of genotype and mutation were 96.0% and 82.5%, respectively, whereas the inconsistent data resulted from a low proportion (<20%) of mixed genotypes or mixed mutations. The mutation ratio in HBV infection patients was as follows: hepatitis B e antigen (HBeAg)-positive infection (0/0.0%) < HBeAg-negative infection (16/4.5%) < HBeAg-positive hepatitis (30/5.5%) < HBeAg-negative hepatitis (36/6.5%). In patients with entecavir therapy, the proportion of mutation at baseline or week 4 in virologic response (VR) group was <4%, whereas in the partial VR group, it was mostly ≥4%. COLD-PCR/FMCA provides a novel tool with high sensitivity, convenience, and practicability for the simultaneous quantification of HBV DNA, genotype, and mutation. It might be used for distinguishing the different phases of HBV infection and predicting VR of CHB patients.

Association of serum lipids with autoantibodies and inflammatory markers in rheumatoid arthritis patients.

BACKGROUND: We studied the relationship between serum lipids and autoantibodies and inflammatory markers in rheumatoid arthritis (RA) patients to explore the effect of serum lipids on the diagnosis and judgment of disease activity in RA patients. METHODS: Serum lipids including TCHO, TG, HDLC and LDLC and anti-CCP, RF, CRP, ESR of RA patients from May 2013 to August 2017 were retrospectively analyzed in the First Affiliated Hospital of Fujian Medical University. RESULTS: With the dilution factor increased, the concentrations of serum lipids and anti-CCP, CRP and RF showed the same downward trend, indicating that the detection methods of the above indicators were reasonable and would not be affected by hyperlipidemia. CRP and ESR levels were negatively correlated with HDLC concentration in male and female RA patients. However, the concentration of anti-CCP and RF were closely related to TG. In all the RA patients and female RA patients, the RF level was negatively correlated with the TG concentration. Moreover, with the TG concentration increased, the proportion of patients with high concentrations of anti-CCP levels decreased. In addition, in male RA patients, anti-CCP and ESR concentration increased with the increase of LDLC. CONCLUSION: The concentrations of HDLC, TG and LDLC were associated with the concentration of anti-CCP, RF, CRP and ESR in RA patients. Therefore, clinical diagnosis of RA and determination of disease activity should consider the impact of the concentration of serum lipids in order to make a reasonable judgment on the diagnosis of the disease.

Red blood cell distribution width: a potential laboratory parameter for monitoring inflammation in rheumatoid arthritis.

Correlation analysis of red blood cell distribution width (RDW) and C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-10 in rheumatoid arthritis (RA) to investigate whether RDW can serve as a potential parameter for indicating inflammation in RA patients. A total of 670 RA patients from October 2014 to April 2016 were enrolled in our study. The white blood cell (WBC), red blood cell (RBC), platelet (PLT), hemoglobin (HGB), RDW, CRP, and ESR in peripheral blood of patients with RA were retrospectively analyzed. The relative expression of TNF-α, IL-6, and IL-10 was detected by RT-qPCR. Correlation analysis between RDW and CRP, ESR, TNF-α, IL-6, and IL-10 in RA was conducted by Microsoft Excel. RDW level was significantly increased in RA patients compared to osteoarthritis (OA) patients (P < 0.001) and healthy donors (HDs) (P < 0.001), and RDW was positively associated with inflammatory markers, such as CRP and ESR. In ROC curve analysis, the area under the curve (AUC) of RDW for the identification of RA was 0.881, with a 95% confidence interval (CI) from 0.864 to 0.898. Moreover, correlation analysis showed that RDW level was positively associated with TNF-α and IL-6, however negatively associated with IL-10. RDW was increased in patients with RA which was associated with inflammation in RA, suggesting that RDW may be a potential auxiliary marker for indicating inflammation process in RA conveniently.

A critical role of transcription factor YY1 in rheumatoid arthritis by regulation of interleukin-6.

Previous studies have revealed a critical role of YY1, a "Yin Yang" transcription factor, in cancer development and progression. However, whether YY1 has any role in rheumatoid arthritis (RA) remains unknown. This study aims to explore the potential role of YY1 in RA pathogenesis. In this study, we found that YY1 was over-expressed in RA patients and CIA mice. Blocking of YY1 action with YY1 shRNA lentivirus ameliorated disease progression in CIA mice. We further analyzed the signaling pathway involved by ingenuity pathway analysis (IPA), results showed IL-6 signaling and JAK/Stat signaling pathway was significantly inhibited by LV-YY1-shRNA treatment. Moreover, we observed that blocking of YY1 reduced IL-6 production and downregulated Th17 population. Finally, we showed YY1 positively regulated IL-6 transcription by binding to the promoter region of the IL-6 gene. In conclusion, YY1 plays a critical role in promoting IL-6 transcription in RA which contribute to the inflammation of RA via stimulation of Th17 differentiation. Thus, YY1 is likely a key molecule involved in the inflammation process of RA. Targeting of YY1 may be a novel therapeutic strategy for RA.

Datasets of YY1 expression in rheumatoid arthritis patients.

The data presented in this article are related to the research article entitled "A critical role of transcription factor YY1 in rheumatoid arthritis by regulation of interleukin-6" (J. Lin, Y. He, J. Chen, Z. Zeng, B. Yang, Q. Ou, 2016) [1]. The article describes YY1 overexpression is specific for RA, but not for SLE, SS, DM or MCTD. In early RA, YY1 expression is also increased. In asymptomatic subjects with RF or ACPA positive who have high risk for developing RA, the YY1 expression is not increased obviously. Moreover, YY1 expression is positively correlated with serum CRP or ESR. In RA patients treated with anti-IL-6R monoclonal Ab tocilizumab, there is no significant difference in YY1 expression after IL-6 blocking therapy.

[A mechanical model of knee joint in sagittal plane].

A sagittal plane model of knee joint based on crossed-four-bar-linkage-based tibiofemoral joint model has been developed using geometric and force equilibrium constraints. The model predicts and explains the movement of contact point on the patella and femur, variation of patellar and patellar tendon angle, variation of patellar mechanism angle and variation of patellofemoral contact force and patellar tendon force. The computed results agree well with the published experimental results.

[Rolling friction: a desing of artificial knee joint].

Resorption and osteolysis of periimplant bones resulting from the wear debris of artificial joint will cause long-term loosening. A new type of rolling knee artificial joint without UHMWPE based on the mechanics of rolling friction is designed for alleviating this problem. Because of low friction force, the resistance of extension and flexion is reduced strikingly and the stress on the interface between prosthesis and bone is reduced evidently. In addition, the bio-toxicity caused by the wear debris of UHMWPE will not occur absolutely. In consequence, the rolling artificial joint can prevent the trend of long-term loosening of the prosthesis efficiently.

[Progress of mechanical model for the study of knee biomechanics].

The mechanical model of knee is a system of constructional elements with specific restrains and material properties, which are deduced from the components of knee based on their functions and junctions. Kinematics and kinetics of knee can be calculated from the mechanical model. A review of some mechanical models of knee are shown in this paper.

The influence of changes in patellar and femoral prosthesis on knee extensor mechanism after TKA.

In order to study the influence of some position changes of prosthesis in patello-femoral joint on knee mechanism after total knee arthroplasty, a three-dimensional model of knee joint including patello-femoral joint has been developed. Changes in position parameters of patellar or femoral prosthesis were simulated. Patellar lateral transition, ratio of patellar tendon force to quadriceps force, and ratio of patello-femoral compressive force to quadriceps force are choosing to response performances of knee extensor mechanics. Results show that patellar thickening, patellar medial rotation, lateral rotation and forward rotation of femoral prosthesis will all increase the patello-femoral compressive force. And patellar thickening, patellar medial rotation, medial rotation and forward rotation of femoral prosthesis will also result in increase of ratio of patellar tendon force to quadriceps. And patellar medial rotation, medial rotation and forward rotation of femoral prosthesis will result in increase of patellar lateral translation.

[Custom design of hip joint prostheses based on X-ray films].

A novel method for the design of hip joint prostheses based on X-ray films is introduced. Only arcs and straight-lines form figures of hip joint prostheses. Because geometrical tolerances of manufacturing hip joint prostheses matching section are usually not strict, hip joint prostheses can be manufactured without CNC machine tool. Three hip joint prostheses for three different femurs were designed through a program which was developed by the present authors. Approximate marrow cavities of these femurs were simulated according to a standard database about femur. These models of femur marrow cavities were used to verify the hip joint prostheses designed. These hip joint prostheses designed were manufactured and implanted into femurs respectively. Experimental results indicate that the novel method for the design of hip joint prostheses is practicable.