How to process synovial fluid samples of gouty arthritis and extract its exosomes for subsequent cytokine analysis.

BACKGROUND: The comparative analysis of ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation for the isolation of exosomes in gouty arthritis synovial fluid (GASF) is rarely reported, and it is not known whether different isolation methods can influence subsequent cytokine analysis. METHODS: GA patients were enrolled during a 1-year period from May 2021 to May 2022. Morphology, particle number, size, purity, protein concentration, and biomarker proteins of GASF-derived exosomes in both extraction methods were observed using transmission electron microscopy, nanoparticle tracer analysis, bicinchoninic acid assay, and Western blotting. An ELISA-based assay platform was used to detect the cytokines in exosomes using Meso Scale Discovery. RESULTS: Thirty-two cases of fresh GASF were taken and randomly divided between the UC group (n = 16) and the PEG group (n = 16). Transmission electron microscopy images and nanoparticle tracer analysis results showed round vesicles measuring 100 nm on average. The protein expressions of TSG101, CD63, and CD81 in exosomes of the 2 groups were measured via Western blotting. The number and protein concentration of GASF-derived exosome particles from the PEG group were significantly higher than that of the UC group (P < .001). However, in the purity estimation, the UC group reflected significantly higher exosomes extractability (P < .01). Expression of IL-6 and IL-8 in the GASF-derived exosomes were higher in the UC group (P < .05), showing a median of 3.31 (interquartile range, IQR: 0.84-13.16) pg/mL, and a median of 2.87 (IQR: 0.56-13.17) pg/mL, respectively; moreover, IL-1ß was mostly undetectable in the PEG group. CONCLUSION: The UC method was found to yield exosomes of a higher purity, albeit at a lower quantity but with more abundant inflammatory cytokines; whereas the opposite was the case for the PEG group. The chemical precipitation method might not be suitable in terms of extracting GASF-derived exosomes for inflammation and immunity studies.

Is Chinese herbal formula (nourishing Yin therapy) effective and well tolerated as an adjunct medication to hydroxychloroquine in the treatment of primary Sjögren's syndrome? A meta-analysis of randomised controlled trials.

BACKGROUND: Previously, data mining methodology was used to identify 71 patented prescriptions in Chinese patent databases, indicating that Yin-nourishing therapy (YNT) may be an adjunct medication to hydroxychloroquine in the treatment of primary Sjögren's syndrome (pSS). The purpose of this study was to investigate effects of the addition of YNT, which includes tonifying liver and kidney therapy (TLKT) and replenishing Qi and nourishing Yin therapy (RQNYT), in the treatment of pSS. METHODS: Fourteen databases (including Chinese, English, Japanese, Korean and Latin databases) were searched to identify randomised controlled trials (RCTs) of YNT plus hydroxychloroquine (YNTPH) versus hydroxychloroquine alone in patients with pSS. Relevant publications up to June 2021 were selected. A meta-analysis and trial sequential analysis (TSA) were performed using Review Manager 5.3, Stata 14.0 and TSA 0.9 software. The quality of identified studies was assessed based on the Cochrane risk of bias tool and GRADE (grading of recommendations, assessment, development and evaluation) criteria. RESULTS: We included five RCTs with a total of 345 participants. Pooled results indicated significant differences in short-term outcomes, which were assessed via European League Against Rheumatism (EULAR) Sjögren's Syndrome Patient Reported Index (ESSPRI), EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI), tear production, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and immunoglobulin G (IgG) results when YNTPH was compared with hydroxychloroquine use alone (p < 0.05). No significant difference in salivary flow rate was reported. The most common side effect reported for both groups was gastrointestinal reaction. Sensitivity analyses suggested that heterogeneity might be ascribed to clinical methodology. Subgroup analyses revealed heterogeneities regarding salivary flow rate were eliminated. TLKT appeared to be better than RQNYT for improving salivary flow rate. TSA only supported changes in ESSPRI, ESSDAI and ESR values. For all studies, the quality of evidence was low. CONCLUSION: YNTPH may be an effective complementary therapy. Current evidence, however, is insufficient for determining whether YNTPH is more effective than hydroxychloroquine alone. Well-designed RCTs are needed to determine the role of YNT in pSS treatment.

Study of the Treatment Effects of Compound Tufuling Granules in Hyperuricemic Rats Using Serum Metabolomics.

The study aimed to investigate the mechanism of the effect of Compound Tufuling Granules (CTG) to lower the serum uric acid level in a rat model of hyperuricemia. The rat model was established by administering hypoxanthine through oral gavage and potassium oxonate through intraperitoneal injection. Rats were divided into the normal group, model group, CTG group, and allopurinol group. Serum uric acid, creatinine, urea nitrogen, and inflammatory cytokine levels were determined in each group. In the model group, ultrahigh performance liquid chromatography-mass spectrometry was used to analyze the metabolic profiles and delineate the action mechanism of CTG; in addition, the orthogonal projection method was used to perform latent structure-discrimination analysis to screen the related metabolites. The results indicated significant differences in the metabolic profiles between the model and normal groups. A total of seven related metabolites were identified through screening in the model group, mainly related to the pathways of bile secretion, pyrimidine, purine, and phenylalanine metabolism, pantothenate and CoA biosynthesis, and pentose and glucuronate interconversions; these related pathways were reversed in the CTG group. In the metabolic networks, uracil and acetyl-coenzyme A were the nodal molecules. In addition, the test results of the evaluation of serum biochemical and inflammatory factors confirmed that CTG had significant effect in reducing the levels of serum uric acid and protecting renal function. These results confirmed that CTG primarily regulated the recruitment of nodal molecules to achieve anti-inflammatory effects, reduced uric acid level, and renal protection.

Compound Tufuling Granules ([characters: see text]) regulate glucose transporter 9 expression in kidney to influence serum uric acid level in hyperuricemia mice.

OBJECTIVE: To explore the effect of Compound Tufuling Granules ([characters: see text], CTG) on regulating glucose transporter 9 (GLUT9) expression in the kidney to influence the uric acid excretion by the kidney and serum uric acid (SUA) level in hyperuricemia mice. METHODS: Sixty Kunming male mice were randomly divided into the control group, model group, benzbromarone group, and CTG high-, middle- and low-dose groups. The yeast extract and uricase inhibition method were used to build hyperuricemia model, and the corresponding drugs were administrated on the 7th day. On the 21st day the 24-h urine was collected, on the 22nd day the blood was collected, the SUA level was detected by uricase colorimetry, and the mRNA and protein expressions of GLUT9 were detected by quantitative real-time polymerase chain reaction and Western blot, respectively. RESULTS: Compared with the model group, the levels of SUA and the mRNA and protein expressions of GLUT9 were significantly decreased, and the fraction excretion of uric acid (FEUA) was significantly increased in the CTG groups and benzbromarone group (all P<0.05). There was no significant difference in the above indicators between the CTG high-dose group and benzbromarone group (P>0.05). SUA is positively related to the GLUT9 mRNA and protein expressions in the kidney (P<0.05 or P<0.01). CONCLUSIONS: CTG can significantly reduce the SUA and increase the FEUA. In addition, CTG can effectively inhibit the mRNA and protein expressions of GLUT9 in the kidney of hyperuricemia mice to inhibit the uric acid re-absorption, promote uric acid excretion and reduce SUA.

Effects of Xie-Zhuo-Chu-Bi-Fang on miR-34a and URAT1 and their relationship in hyperuricemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xie-Zhuo-Chu-Bi-Fang (XZCBF) is an empirical formula that was developed based on the principles of traditional Chinese medicine, for the therapeutic purpose of treating hyperuricemia. XZCBF has been clinically utilized in the Department of Traditional Chinese Medicine at General Hospital of Guangzhou Military Command of PLA for many years and has exhibited favorable efficacy. The aim of the study is to evaluate the effects of XZCBF on the expression of uric acid transporter 1 (URAT1) and miR-34a in hyperuricemic mice and to determine, the correlation between the two expression levels. MATERIALS AND METHODS: A hyperuricemic animal model was created by administering adenine and allantoxanic acid potassium salt to mice. The blood uric acid levels were measured in these model mice after treatment with XZCBF for 15 days. The potential targets of miR-34a were screened. The expression levels of miR-34a and URAT1 in the renal tissues collected from the model mice were determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and their correlation was further established by immunohistochemistry and in situ hybridization. RESULTS: The uric acid levels in the model mice were significantly higher than those in the blank controls (P<0.05). These levels were significantly lower in the three groups receiving different doses of XZCBF (P<0.05), which was, in agreement with the downregulation of URAT1 and the upregulation of miR-34a in each group. The mRNA expression level of URAT1 was positively correlated with the concentration of uric acid but, negatively correlated with the expression level of miR-34a. CONCLUSIONS: The ability of XZCBF to facilitate the excretion of uric acid and to lower its level in the model group was mediated by the upregulation of miR-34a and the inhibition of URAT1 mRNA expression, which suggests that XZCBF could be an option for the treatment of hyperuricemia in mice.

[Study of computed tomographic dacryocystography used in endoscopic intranasal dacryocystorhinostomy preoperatively].

OBJECTIVE: To study computed tomographic dacryocystography used in endoscopic intranasal dacryocystorhinostomy preoperatively. METHOD: Ten volunteers and twelve patients with dacryocystitis were undergone the coronary and axial computed tomographic dacryocystography. RESULT: According to the anatomic relationship between anterior ethmoid sinus and fossa of lacrimal sac, the cells of anterior ethmoid sinus were classified into three typies: the result was that type I accounted for 12 sides (27.3%), type II 18 sides (40.9%), type III 14 sides (31.8%) by analysing the data of axial dacryocystographies; the sites of obstruction were connection of the lacrimal sac with nasolacrimal duct, the form of lacrimal sac of 11 patients was normal or enlarged lacrimal sacs, bilateral cricatrized lacrimal sacs were found in one patient by analysing the data of coronary dacryocystographies. CONCLUSION: Computed tomographic dacryocystography preoperatively can provide significant clinical guidance for the endoscopic transnasal dacryocystorhinostomy in locating the site of bone-opening.