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To determine the effects of berberine (BBR) on the activation of toll-like receptor 4 (TLR4), nuclear factor (NF)κB (NF-κB) signaling and NLRP3 inflammasome in patients with gout.Peripheral blood mononuclear cells (PBMCs) were obtained from 24 acute (AP) and 41 non-acute (NAP) phases of primary gout patients, respectively, as well as 30 healthy controls (HC). TLR4, NF-κB (p65), NLRP3, apoptosis-associated specklike protein containing a CARD (PYCARD), cysteinyl aspartate specific proteinase-1 (CASP1), and interleukin-1β (IL-1β) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction. The protein levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB (p50/65), inhibitor of kappa B kinase α/β (IKKα/β), NF-κB inhibitor α (IKBα), phospho-IKKα/β (p-IKKα/β), NLRP3, PYCARD, and CASP1 were monitored by Western blotting. Serum IL-1β protein level was measured using enzyme-linked immunosorbent assay (ELISA). In addition, PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line (THP-1) were stimulated using monosodium urate (MSU, 100 µg/mL), 0.1% dimethyl sulfoxide, 25 µmol/L BBR, and 10, 25, and 50 µmol/L BBR+100 µg/mL MSU for different time periods. The protein levels of IL-1β and IL-18 in cell culture supernatants was measured by ELISA, and the protein expressions of TLR4, MyD88, NF-κB (p50/p65), IKKα/β, I κBβ, p-IKKα/β, NLRP3, PYCARD, and CASP1 in macrophages were analyzed by Western blotting.(1) TLR4, NF-κB (p65), PYCARD, CASP1, and IL-1β mRNA levels in PBMCs were significantly higher in the AP group than in the HC group (P0.05). The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group (P0.05, P0.01). (2) The protein levels of TLR4, IKKβ, MyD88, NF-κB, p-IKKα/β, PYCARD, and CASP1 in PBMCs were significantly higher, and those of IκBα, IKKα, and NLRP3 were found to be significantly lower in the AP group than in the HC group (P0.05 or P0.01). (3) The serum IL-1β protein levels were significantly higher in the AP and NAP groups than in the HC group (P0.01). (4) The IL-1β protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 µmoL/L BBR groups compared with that in the MSU group (P0.01). (5) The protein levels of IL-1β and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group (P0.01). (6) The protein levels of TLR4, MyD88, NF-κB (p50, p65, p105), IKKα/β, p-IκBα, p-IKKα/β, PYCARD, and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group (P0.05 or P0.01).Activation of TLR4-NFκB signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation. BBR ameliorates gouty inflammation, which is mechanistically associated with its regulation of TLR4-NF-κB signaling and NLRP3 inflammasome expression.

Enterocytozoon bieneusi is a zoonotic fungal pathogen with a high degree of host diversity that can parasitize many animals, including humans. Pigs may play an important role in the epidemiology of E. bieneusi as reservoir hosts. Nevertheless, the genotypes of E. bieneusi in pigs in China remain poorly understood. The aim of this study was to determine the prevalence of E. bieneusi infection amongst pigs raised on farms from four cities of Hainan Province, using nested polymerase chain reaction (PCR) of the partial small subunit of the ribosomal RNA gene, and to identify genotypes of E. bieneusi isolates based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Among 188 stool samples, E. bieneusi was detected in 46.8% (88/188). Eight genotypes including four known (EbpA, CS-4, MJ14, and CHG19) and four novel (HNP-I - HNP-IV) genotypes were identified. Using phylogenetic analysis, genotypes EbpA, CS4, CHG19, HNP-III, and HNP-IV were clustered into zoonotic Group 1, while the remaining three genotypes (MJ14, HNP-I, and HNP-II) clustered into Group 10. The high prevalence of zoonotic genotypes of E. bieneusi among pigs suggests that pig farming is a potential source of human infection. Additionally, this is the first identification of genotypes in Group 10 in pigs indicating unique epidemic features of E. bieneusi in pigs in Hainan Province, the southernmost part of China.Détection moléculaire d’Enterocytozoon bieneusi chez les porcs d’élevage dans la province de Hainan en Chine : taux d’infection, répartition des génotypes et potentiel zoonotique.Enterocytozoon bieneusi est un pathogène fongique zoonotique avec une grande diversité d’hôte qui peut parasiter de nombreux animaux, y compris les humains. Les porcs peuvent jouer un rôle important dans l’épidémiologie d’E. bieneusi en tant qu’hôtes réservoirs. Néanmoins, les génotypes d’E. bieneusi chez le porc en Chine restent mal connus. Le but de cette étude était de déterminer la prévalence de l’infection par E. bieneusi chez les porcs élevés dans des fermes de quatre villes de la province de Hainan, en utilisant la réaction en chaîne par polymérase emboîtée (PCR) de la petite sous-unité partielle du gène de l’ARN ribosomal et de identifier les génotypes des isolats d’E. bieneusi sur la base d’une analyse de séquence de la région des espaceurs internes transcrits ribosomiques (ITS). Sur 188 échantillons de selles, E. bieneusi a été détecté dans 46,8 % (88/188). Huit génotypes, dont quatre génotypes connus (EbpA, CS-4, MJ14 et CHG19) et quatre génotypes nouveaux (HNP-I à IV), ont été identifiés. Dans une analyse phylogénétique, les génotypes EbpA, CS4, CHG19, HNP-III et HNP-IV étaient regroupés dans le groupe zoonotique 1, tandis que les trois génotypes restants (MJ14, HNP-I et HNP-II) étaient regroupés dans le groupe 10. La prévalence élevée des génotypes zoonotiques d’E. bieneusi chez les porcs suggère que l’élevage porcin est une source potentielle d’infection humaine. De plus, il s’agit de la première identification de génotypes du groupe 10 chez les porcs, indiquant des caractéristiques épidémiques uniques d’E. bieneusi chez les porcs dans la province de Hainan, la partie la plus méridionale de la Chine.

To analyze the levels of oxidized low density lipoprotein (ox-LDL) and inflammatory cytokines in the plasma of gout patients. The levels of ox-LDL, hypersensitive C-reactive protein (hs-CRP), interleukin-1β, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured in the plasma of 41 gout patients [28 in acute phase episode, 13 in intermittent phase (IP)], and in 40 healthy controls. The relationship between ox-LDL and inflammation was also explored by measuring the levels of several pro-inflammatory cytokines in the plasma. The plasma levels of ox-LDL, hs-CRP, IL-6 and TNF-α were significantly increased in patients with gout in the acute phase compared to those in the IP group and healthy controls (P

Gout patients have a high incidence of atherosclerotic coronary heart disease. Low serum paraoxonase (PON) activity is considered a risk factor for atherosclerosis. The relationships among paraoxonase-1 (PON1) activity, oxidative stress parameters, and atherosclerosis in gout is not known. Therefore, we determined the plasma levels of malondialdehyde (MDA), oxidized low-density lipoprotein (Ox-LDL), and activities of PON1/superoxide dismutase (SOD) activities in 49 gout patients (mean age 44.2 ± 7.0 years) and 42 healthy, age-matched controls (mean age 45.0 ± 9.3 years). PON1 was measured spectrophotometrically, MDA by thiobarbituric acid method, SOD by Griess reaction, and Ox-LDL by sandwich ELISA. Lipid and other biochemical parameters were determined by routine laboratory methods. In gout patients, PON1/SOD activities and MDA/Ox-LDL levels were 131.3 ± 25.3/75.3 ± 28.9 kU l(-1) and 6.12 ± 1.67 nmol ml(-1)/690.1 ± 180.2 μg l(-1), respectively. In controls, these were 172.5 ± 27.8/94.0 ± 26.3 kU l(-1) and 4.10 ± 1.25 nmol ml(-1)/452.3 ± 152.1 μg l(-1), respectively. Thus, in gout patients, there was a significant decrease in PON1 (P < 0.01) and SOD (P < 0.05) activities, and an increase in MDA (P < 0.01) and Ox-LDL (P < 0.01) levels compared with controls. PON1 activity correlated positively with SOD (P < 0.05), and negatively with MDA (P < 0.01) and Ox-LDL (P < 0.01). These results suggest that gout patients were in a state of oxidative stress and the protective effects of HDL against atherosclerosis maybe dependent on PON1 activity. These findings may explain in part the reported increase in cardiovascular mortality in gout patients.

To determine the expression level and role of PYCARD [PYRIN-PAAD-DAPIN domain (PYD) and a C-terminal caspase recruitment domain (CARD), PYCARD] gene and its transcript variant mRNA in peripheral blood mononuclear cells (PBMCs) of patients with primary gout (PG).PYCARD gene and its transcript variant mRNA were measured using reverse transcription-polymerase chain reaction (RT-PCR) in PBMCs. The expression of PYCARD gene and PYCARD-1,-2 mRNA in PBMCs was compared between the patients with acute phase PG (APPG) (n=44), non-acute phase PG (NAPPG) (n= 51) and healthy controls (HC) (n=87). PYCARD and NF-kappaB (p105/p50) protein expressions were measured using Western blot in the PBMCs of participants in the PG and HC groups. Routine blood tests and blood uric acid test were undertaken in all participants. Differences in the indicators were examined among the three groups. Correlations between the expression of PYCARD gene and PYCARD-1,-2 mRNA and other indicators were analyzed.The expression level of PYCARD gene, PYCARD-1,-2 mRNA was significantly higher in the APPG and NAPPG group than in the HC group (P0.01). The NAPPG group had significantly higher levels of PYCARD gene transcript variant 2x mRNA and 2y mRNA in the HC and APPG groups (P0.05). The expression of PYCARD and NF-kappaB (p105/p50) protein was significantly higher in the PG group compared with the HC group [(4.900 +/- 1.324) vs. (3.975 +/- 0.210) and (0.263 +/- 0.106) vs. (0.127 +/- 0.008), respectively P0.05]. The expression level of PYCARD-2 mRNA and granulocyte were positively correlated in the NAPPG group.Abnormal expression of PYCARD gene and its transcript variant and PYCARD protein in PG patients suggests that PYCARD gene and its transcript variant may play an important role in regulating the inflammatory response of PG patients.

To identify the proapoptotic effects of Gynostemma pentaphyllum Makino (GpM) on human hepatoma cells. The effects of GpM on the cell apoptosis of human hepatoma cell line Huh-7 was assessed by flow cytomety. The expression of Bcl-2, Bcl-XL, Bax and Bad molecules in hepatoma cells treated with GpM was detected by Western blot. After treatment with 20 mg/mL GpM for 24 h, 56% of Huh-7 cells were undergoing apoptosis, while cell death was only observed in 12% of human fibroblast cells treated with GpM. Western blot demonstrated that, Bcl-2 was markedly decreased in Huh-7 cells treated with GpM. Whereas, Bax was significantly up-regulated in Huh-7 cells treated with GpM. Treatment of human hepatoma cells with GpM induced apoptosis through the down-regulation of Bcl-2, and up-regulation of Bax.

To understand the difference in clinical indicators of gout patients of different Chinese medical syndromes and its clinical significance.Form November 2011 to December 2012, syndrome typed were 257 male gout in-/outpatients from Affiliated Hospital of Chuanbei Medical College. Another 50 healthy male subjects were recruited as the control. Their clinical and laboratory data were collected. All were excluded from infections and other inflammatory diseases.Four syndrome types existed in gout patients, i.e., intermingled phlegm-stasis blood syndrome (IPSBS), obstruction of dampness and heat syndrome (ODHS), Pi-deficiency induced dampness syndrome (PDIDS), qi-blood deficiency syndrome (QBDS). Of them, 53 acute phase gout patients suffered from IPSBS, 41 from ODHS, 25 from QBDS, and 17 from PDIDS; 41 non-acute phase gout patients suffered from QBDS, 40 from PDIDS, 24 from ODHS, and 16 from IPSBS. Statistical analysis of clinical data showed that, when compared with the normal control group, there was statistical difference in blood routines (WBC, GR, LY, MO) and blood biochemical indices (UA, Ur, Cr, ALT, AST, ALB, GLOB, TG, HDL-C, VLDL-C, apoA, apoB100) of gout patients of different syndromes (P0.05, P0.01). There was also statistical difference or correlation among different syndromes (P0.05).In the acute phase gout patients, IPSBS and ODHS were dominated, while in the non-acute phase gout patients, QBDS and PDIDS were often seen. In patients of IPSBS and ODHS, inflammation and immune response were more obvious, indicating that better efficacy might be achieved by clearing heat and removing blood stasis associated anti-inflammatory and immune regulation therapies. In patients of QBDS and PDIDS, impaired renal functions were more significant, indicating that better efficacy might be achieved by invigorating Pi and tonifying Shen dominated treatment.

To explore the oxidative mechanism of uric acid (UA) induced CRP expression in human umbilical vein endothelial cells.Different concentrations of UA (0 mg/dL, 4 mg/dL, 8 mg/dL, 12 mg/dL, 16 mg/dl) were incubated 12 h with HUVECs, and HUVECs were stimulated with 12 mg/dl. UA for different times (6 h, 12 h, 24 h, 48 h). CRP mRNA and protein expression were determined by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively; the effects of uric acid on the intracellular reactive oxygen species (ROS) production in HUVECs were measured by fluorescence microscope and flow cytometric analysis using a 2', 7'-Dichlorofluorescin diacetate (DCF-DA) fluorescence probe. The effects of N-acetyl cysteine (NAC) on UA-induced levels of ROS, mRNA and protein of CRP in HUVECs were also observed.The results demonstrated that UA could significantly increase the mRNA and protein expression of CRP in HUVECs in time- and concentration-dependent manners. HUVECs were stimulated with 12 mg/dL UA at 6 h, mRNA and protein levels of CRP significantly higher than that of control level (P0.05), reached a peak at 12 h (P0. 01). NAC reduced UA-induced levels of ROS, mRNA and protein of CRP in HUVECs compared with those of 12 mg/dL UA induced group(P0. 05).Uric acid significantly increased mRNA and protein expression of CRP in HUVECs in time- and concentration-dependent manners. Its mechanism may be associated with uric acid induced increasing of ROS levels in endothelial cells, which suggested that the uric acid mediated oxidative stress and inflammation may be involved in the injury of endothelial cells.

Gout is the most common autoinflammatory arthritis characterized by elevated serum urate and recurrent attacks of intra-articular crystal deposition of monosodium urate (MSU) in tissues. The pathogenesis of gout has not been fully determined, although certain genetic factors are involved in the development of gout. Accumulated data suggested that MSU crystal-induced inflammation is a paradigm of innate immunity. As Toll-like receptors (TLRs) are the underlying mechanisms of the innate immune response, the present study aimed to investigate whether TLR2 polymorphisms are associated with gout. Two single-nucleotide polymorphisms (Arg677Trp and Arg753Gln, rs5743708) in TLR2 were genotyped by polymerase chain reaction-restriction fragment length polymorphism and the -196 to -174 del polymorphism was investigated using the allele-specific polymerase chain reaction in 431 individuals (215 patients with gout and 216 healthy controls). TLR2 Arg677Trp and Arg753Gln genotyping indicated that all the positive samples were of the wild-type genotype. No significant differences in genotype (χ2=1.686, P=0.430) and allele (χ2=1.430, P=0.232) frequencies of the -196 to -174 del polymorphism between the patients with gout and the control groups was observed. Our results suggested that the TLR2 Arg677Trp, Arg753Gln and the -196 to -174 del polymorphisms were not associated with susceptibility to primary gouty arthritis.

To investigate the production of membrane-expressed urokinase-type plasminogen activator receptor (muPAR) and cytoplasmic-expressed uPAR (cuPAR) in synovial tissue from patients with rheumatoid arthritis (RA) and analyze their relationship with the severity of synovial inflammation in RA.MuPAR and cuPAR were measured with indirect immunofluorescence (IIF) and immunoperoxidase histochemical analysis of the synovial tissue sections from 18 patients with RA, 10 patients with osteoarthritis (OA) and 4 healthy subjects. The association of uPAR expression with the severity of synovitis in RA was then analyzed.MuPAR positive cells were detected in approximately (66.0+/-9.4)% of the RA synovial cells, distributed predominantly in vascular endothelial cells and fibroblast cells. While cuPAR positive cells were found in about (61.0+/-5.8)% of the RA synovial cells, including subsynovial and interstitial macrophage-like cells, mononuclear leukocytes and fibroblast cells. Both the muPAR and cuPAR expression were much more increased in RA synovial tissue than those in OA synovial tissue. Furthermore, the number of muPAR (r=0.672, P0.01) and cuPAR (r=0.649, P0.01) positive cells in synovial tissue was also found to be correlated significantly with the severity of synovial inflammation in RA patients.The up-regulated expression of muPAR in synovial vascular endothelial cells suggests an important role of this molecule in angiogenesis in RA and the increased production of cuPAR in synovial inflammatory cells indicates the involvement of cuPAR in the inflammatory process in RA.

To investigate the change of serum tumor necrosis factor-alpha (TNFalpha) and endothelin (ET) levels and their clinical significance in patients with bee sting poisoning.TNFalpha and ET were measured in 97 patients with bee sting poisoning and the results were compared with those in 35 healthy controls. The 97 patients were divided into three groups i.e. mild poisoning group, moderate poisoning group and severe poisoning group and they were also divided according to prognosis into death group and survival group.The levels of serum TNFalpha (1.36 +/- 0.37) microg/L and ET (55.20 +/- 12.60) ng/L in the slight poisoning group increased a little, but they were not significantly different from those in the controls (1.09 +/- 0.25) microg/L and (50.90 +/- 8.80) ng/L (P0.05). In the moderate and severe poisoning groups the levels of serum TNFalpha (2.82 +/- 0.79) microg/L, (4.02 +/- 0.93) microg/L and plasma ET (139.30 +/- 58.80) ng/L, (210.50 +/- 86.30) ng/L were significantly higher than those in the controls (P0.01). The levels in the severe poisoning group were much higher than those in the moderate poisoning group and the latter much higher than those in the controls. The levels of serum TNFalpha (4.53 +/- 0.89) microg/L and plasma ET (267.50 +/- 98.70) ng/L in the death group were much higher than those in the survival group (2.40 +/- 0.82) microg/L, (107.60 +/- 57.90) ng/L (P0.01). The levels of serum TNFalpha were positively correlated with those of plasma ET (P0.01).TNFalpha and ET may be involved in the pathogenesis of bee sting poisoning. It is shown that the higher the levels of serum TNFalpha and plasma ET, the severer degree of the poisoning and the higher fatality rate. Serum TNFalpha and plasma ET levels may be important clinical index in judging the patients, condition and their prognosis in bee sting poisoning. It was an important clinical significance, that may be useful for medicating with antagonist and inhibitor against TNFalpha and ET to prevention and depress death rate in the patients with bee sting poisoning.

To investigate the correlation between the change of plasma endothelin (ET) and the occurrence and progress of multiple organ dysfunction syndrome (MODS) caused by fish gall bladder poisoning.Plasma ET was measured in 85 patients with fish gall bladder poisoning and 30 healthy controls and comparison of the levels of plasma ET was carried out between patients groups with different severity of fish gall bladder poisoning. The relationship between plasma ET levels and enzymology as well as some biochemistry indexes in patients with fish gall bladder poisoning was then analyzed.Plasma ET in patients with fish gall bladder poisoning (150.5 +/- 90.5) ng/L was much higher than that in controls (52.8 +/- 9.3) ng/L. The concentration of plasma ET in slight poisoning group (61.2 +/- 13.6) ng/L increased a little, but it was not significantly different with that in controls (P0.05). The concentration of plasma ET was significantly higher in moderate poisoning group and severe poisoning group (162.3 +/- 82.3) ng/L and (232.6 +/- 92.7) ng/L as compared with that in the controls (P0.01), with the severe poisoning group much higher than the moderate poisoning group and the latter much higher than the normal. At the same time, plasma ET in MODS group and non-MODS group (210.2 +/- 98.7) ng/L and (126.3 +/- 68.3) ng/L were both notably higher than that in the controls. Furthermore plasma ET in the MODS group was much higher than that in the non-MODS (P0.01). It was also found that a positive correlation existed between plasma ET and creatine kinase-MB, cardiac troponin I, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and serum creatinine (P0.01) and a negative correlation between plasma ET and endogenous creatinine clearance rate (P0.01).Plasma ET may be involved in the process of fish gall ladder poisoning and later in the progress of MODS. It was shown that the higher the level of plasma ET, the severer the degree of poisoning and the higher the fatality rate. So monitoring plasma ET level dynamically may be an important clinical index in judging the patients condition and their prognosis in fish gall bladder poisoning. Simultaneously it is useful for earlier diagnosis of fish gall bladder poisoning and alertness of MODS caused by it as well as for proper management with antagonist and inhibitor against plasma ET for prevention and cure of fish gall bladder poisoning.

To study the pathogenic effect of hepatitis G virus (HGV) infection on hepatic failure.Using the RT-PCR and EIA techniques to detect HGV RNA and anti-HGV in sera of hepatic failure patients and compare them with their liver function and mortality rates.There was no significant difference about the positive rates of HGV among acute hepatic failure, subacute hepatic failure and chronic hepatic failure groups (X(2)=2.54, P0.05). The level of ALT in HGV-positive group was slightly lower than that in HGV-negative group. The concentration of bilirubin and globulin was higher in HGV-positive group than HGV-negative group, and the concentration of albumin in HGV-positive group was significantly lower than that in HGV-negative group (t=2.59, P0.05). The mortality rate in HGV-positive group was significantly lower than that in HGV-negative group (X(2)=4.68, 0.01P0.05).The virulence of HGV is mild, and the HGV infection does not aggravate hepatic failure.