Affinity Chromatography Purification of Recombinant Human Interleukin-6 from Its Fusion Protein with GST

Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.

C-reactive protein,procalcitonin,interleukin-6,vascular endothelial growth factor and oxidative metabolites in diagnosis of infection and staging in patients with gastric cancer

AIM:The current study was to determine the serum/pLasma levels of VEGF,IL-6,malondialdehyde (MDA),nitric oxide (NO),PCT and CRP in gastric carcinoma and correlation with the stages of the disease and accompanying infection. METHODS:We examined the levels of serum VEGF,IL-6, PCT,CRP and plasma MDA,NO in 42 preoperative gastric cancer patients and 23 healthy subjects.There were infection anamneses that had no definite origin in 19 cancer patients. RESULTS:The VEGF levels (mean±SD; pg/mL) were 478.05±178.29 and 473.85±131.24 in gastric cancer patients with and without infection,respectively,and these values were not significantly different (P>0.05).The levels of VEGF, CRP,PCT,It-6,MDA and NO in cancer patients were significantly higher than those in healthy controls and the levels of CRP,PCT,It-6,MDA and NO were statistically increased in infection group when compared with non- infection group (P<0.001). CONCLUSION:Although serum VEGF concentrations were increased in gastric cancer,this increase might not be related to infection.CRP,PCT,IL-6,MDA and NO have obvious drawbacks in the diagnosis of infections in cancer patients. These markers may not help to identify infections in the primary evaluation of cancer patients and hence to avoid unnecessary antibiotic treatments as well as hospitalization. According to the results of this study,IL-6,MDA,NO and especially VEGF can be used as useful parameters to diagnose and grade gastric cancer.

Fra-1 protooncogene regulates IL-6 expression in macrophages and promotes the generation of M2d macrophages

The tumor microenvironment （TME） plays a prominent role in the growth of tumor cells. As the major inflammatory component of the TME, M2d macrophages are educated by the TME such that they adopt an immunosnppressive role that promotes tumor metastasis and progression. Fra-1 forms activator protein-1 heterodimers with Jun partners and drives gene transcription. Fra-1 is thought to drastically induce tumorigenesis and progression. However, the functional role of Fra-1 in the generation of M2d macrophages is poorly understood to date. Here, we demonstrate that 4T1 mammary carcinoma cells, when co-cultured with RAW264.7 macrophage cells, skew the RAW264.7 macrophage cell differentiation into M2d macrophages. The 4T1 cells stimulate de novo overexpression of Fra-1 in RAW264.7 cells, and then Fra-1 binds to the interleukin 6 （IL-6） promoter to increase the production of the cytokine IL-6 in RAW264.7 cells. IL-6 acts in an autocrine fashion to skew RAW264.7 macrophage cell differentiation into M2d macrophages. These findings open new insights into how to reverse M2d macrophage-induced immune tolerance to improve the efficacy of immunotherapeutic approaches.

Interleukin-6 and its soluble receptor in patients with liver cirrhosis and hepatocellular carcinoma

AIM： To evaluate the immunohistochemical localization of interleukin-6 （IL-6） and IL-6 receptor （IL-6R） on tumor tissue specimens from patients with hepatocellular carcinoma （HCC） and the serum levels of IL-6 and sIL-6R in a group of patients with HCC as well as liver cirrhosis （LC） in a group of patients with LC alone and in a control group. METHODS： Three groups of subjects were studied： group Ⅰ （n =83） suffering from HCC and LC, group Ⅱ （n = 72） suffering from LC alone and group Ⅲ （n =42） as healthy controls. All patients had hepatitis C virus infection. Serum IL-6 and IL-6R levels were determined using a commercially available ELISA kit. Immunohistochemistry was performed using the streptavidin-biotin complex and rabbit polyclonal antibodies against IL-6 and IL-6R. RESULTS： Immunohistochemistry analysis showed a medium to strong cytoplasmic and membrane reactivity for IL-6 and IL-6R respectively, in at least 40% of cases of HCC, whereas liver cirrhosis patients and controls were negative for IL-6 or showed a very mild and focal dot-like cytoplasmic reaction for IL-6R. Serum IL-6 levels in HCC group were significantly higher than those in LC and control groups （P〈 0.0001）. There was no significant difference in sIL-6R concentrations among 3 groups. When the patients with HCC were divided into groups according to Okuda＇s classification, a significant serum increase of IL-6 and slL-6R level was observed from stage Ⅰ to stage Ⅲ （P〈0.02, P〈0.0005）. When HCC and LC patients were divided into 3 classes of cirrhosis severity according to Child-Pugh, values in HCC patients were significantly higher than those in LC patients for each corresponding class （P〈 0.01）. CONCLUSION： IL-6 serum levels in HCC patients are higher than those in LC patients and controls, suggesting an increased production of this cytokine by neoplastic cells, sIL-6R values are similar in all groups, increasing only in stage III HCC patients. These data suggest that they have a closer relationship with the neoplastic mass rather than with the residual functioning hepatic mass.

Interleukin-6, desmosome and tight junction protein expression levels in reflux esophagitis-affected mucosa

AIM： To investigate the correlation between the expression levels of interleukin （IL）-6 and proteins in tight junctions （TJs） in the esophageal mucosa of rats modeling different types of reflux esophagitis （RE）, and the ability of aluminum phosphate to protect against RE-induced mucosal damage via these proteins. METHODS： Male SPF Wistar rats aged 56 d were divided randomly into acid RE, alkaline RE, mixed RE, and control groups. Various surgical procedures were performed to establish rat models of acid RE. At 14 d after the procedure, some of the rats started aluminum phosphate treatment. Transmission electron microscopy （TEM） was used to observe the morphological features of TJs and desmosomes in the esophageal epithelium. Immunohistochemical methods and Western blotting were used to measure expression of claudin 1, occludin, ZO-1, JAM-l, DSG-1 and IL-6; reverse transcription polymerase chain reaction （RT- PCR） was used to measure expression of mRNA of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6. RESULTS： At day 14 alter the procedures, an RE model was established in all subsequently sacrificed rats of groups A, B and C. By both gross and microscopic observation, the mucosa was damaged and thickened as the disease progressed. With TEM observation, a widened intercellular space was noticed, with significantly fewer desmosomes. Immunohistochemistry showed significantly higher levels of all proteins in all RE models compared to control rats at 3 d after operation （65.5% ± 25.6% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. At 14 d after operation, along with continuing hyperplasia in the basal layer, the expression of TJ proteins in individual cells gradually decreased （12.4% ± 2.1% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. Western blottings and RT-PCR showed a directly proportional increase in IL-6 levels in relation to TJ proteins, as compared to controls （0.878 ± 0.024 vs 0.205 ± 0.021 and 0.898±0.022 vs 0.205 ± 0.021, P 〈 0.05, respectively）. Upon treatment with aluminum phosphate, however, these protein levels were restored to normal gradually over 30-60 d in rats with acid RE （30.4% ± 2.1% vs 20.5% ± 2.1%, P 〉 0.05, treated vs untreated, respectively）. These levels increased in the rat with alkaline RE, and this increase was accompanied by continued hyperplasia in comparison with controls （85.5% ± 25.6% vs 20.5% ± 2.1%, P 〈 0.05, respectively）. Furthermore, the expression of TJ proteins was not correlated significantly with that of IL-6 in this group. CONCLUSION： These findings indicate that TJ proteins are highly expressed as an early molecular event involved in RE development, and that IL-6 is an inflammatory factor in this process,

Glutamine-supplemented total parenteral nutrition attenuates plasma interleukin-6 in surgical patients with lower disease severity

AIM： To evaluate whether the effect of Gin dipeptideenriched total parenteral nutrition （TPN） on postoperative cytokine alteration depended on the disease severity of surgical patients. METHODS： Forty-eight patients with major abdominal surgery were allocated to two groups to receive isonitrogenous （0.228 g nitrogen/kg per d） and isocaloric （30 kcal/kg per d） TPN for 6 d. Control group （Cony） using conventional TPN solution received 1.5 g amino acids/kg per day, whereas the test group received 0.972 g amino acids/kg per day and 0.417 g L-alanyI-L-glutamine （Ala-GIn）/kg per day. Blood samples were collected on d 1 and d 6 postoperatively for plasma interleukin （IL）-2, IL-6, IL-8, and interferon （IFN）-γ analysis. RESULTS： Plasma IL-2 and IFN-γ were not detectable. IL-6 concentrations were significantly lower on the 6^th postoperative day in the Ala-GIn group than those in the Cony group in patients with APACHE Ⅱ≤6, whereas no difference was noted in patients with APACHE Ⅱ〉6. There was no difference in IL-8 levels between the two groups. No difference in cumulative nitrogen balance was observed on d 2-5 after the operation between the two groups （Ala-GIn -3.2±1.6 g vs Cony -6.5±2.7 g）. A significant inverse correlation was noted between plasma IL-6 levels and cumulative nitrogen balance postoperatively in the Ala-GIn group, whereas no such correlation was observed in the Conv group. CONCLUSION： TPN supplemented with Gin dipeptide had no effect on plasma IL-8 levels after surgery. However, Gin supplementation had a beneficial effect on decreasing systemic IL-6 production after surgery in patients with low admission illness severity, and lower plasma IL-6 may improve nitrogen balance in patients with abdominal surgery when Gin was administered.

INTERLEUKIN-6 PROTECTS ANNULUS FIBROSUS CELL FROM APOPTOSIS INDUCED BY INTERLEUKIN-1β IN VITRO

Objective To investigate the effect of interleukin-6 （ IL-6 ） on the apoptosis of annulus fibrosus （AF） cell induced by intedeukin-1β （IL-1β）. Methods Cultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1β, 10 ng/mL IL-1β and Z-VAD-FMK （ a caspase-9 inhibitor）, 10 ng/mL IL-1β and 10 ng/mL IL-6, 10 ng/mL IL-1β and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry. Results The apoptosis rates of cells in group 1 to 6 were 2.67% ± 1.08%, 2.71% ± 0.53%, 20. 37% ± 1.57 %, 11.34% ± 0.67 %, 18.17 % ± 0.74%, and 9.42 % ± 1.08 %, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 （ P = 0. 001, P =0. 172, and P =0. 001, respectively）. Positive rates of caspase-3 in group 5 （ 12. 35% ±0.64% ） and 6 （9.26% ±0. 36% ） were significantly lower than group 3 （ 17.14% ±0. 72% ; P =0. 001 and P 〈0.001, respectively）. And positive rates of caspase-9 in group 5 （ 15.13% ± 1.45% ） and 6 （ 10.17% ± 2.50% ） were significantly lower than group 3 （ 19.4% ±0.98% ; P =0. 014 and P =0. 004, respectively）. But there was not obvious change of caspase-8 activity after IL-6 was added. Conclusion IL-6 is capable of protecting AF cells from IL-1β induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Serum Levels of Interleukin-1 Beta, Interleukin-6 and Melatonin over Summer and Winter in Kidney Deficiency Syndrome in Bizheng Rats

Objective To observe the seasonal changes in serum levels of interleukin-1 beta(IL-1β), interleukin-6(IL-6) and melatonin(MT) in Bizheng rat model, and explore the relationship between MT and the pathogenesis of rheumatoid arthritis. Methods One hundred and sixty Sprague-Dawley rats were randomly divided into four groups in summer(n=80) and winter(n=80) respectively: normal group, collagen-induced arthritis(CIA) model group, operation group, and sham-operation group(n=20 in each group). The CIA model group was injected with collagen emulsion at the base of the tail to induce arthritis. The rats in the operation group received pineal gland resection, and 7 days after the first operation, underwent testectomy or oophorectomy. The rats in the sham-operation group were operated to ligature the sagittal sinus, without extracting the pineal gland. After the operations, the operation group and the sham-operation group both were immunized as the CIA group was. The serum levels of IL-1β, IL-6 and MT in different groups were measured by radioimmunoassay. Results Compared with the normal group, the serum levels of IL-1β and IL-6 increased in the CIA model, operation, and sham-operation groups both in summer and in winter(IL-1β in summer, P=0.008, P<0.01, P=0.012; IL-1β in winter, P=0.019, P<0.01, P=0.027; IL-6 in summer, P=0.028, P<0.01, P=0.024; IL-6 in winter, P=0.006, P<0.01, P=0.008). In the operation group, the serum levels of IL-1β and IL-6 in winter were higher than in summer, but with no statistically significant differences(P=0.844, 0.679). Compared with the normal group, the serum level of MT significantly increased in summer and winter in both the CIA model group(P=0.002, 0.008) and the sham-operation group(P=0.003, 0.007), while significantly decreased in the operation group(P=0.023, 0.003). There was no significant difference in MT level in the operation group between summer and winter(P=0.947). Conclusions The increase of serum levels of IL-1β and IL-6 may exacerbate the inflammatoryreaction and cause a more severe condition in the rheumatoid arthritis. The concentrations of IL-1β, IL-6, and MT correspond with the change of seasons, confirming that there are connections between nature and human body.

Transcriptome of intraperitoneal organs of starry flounder Platichthys stellatus challenged by Edwardsiella ictaluri JCM1680

Platichthys stellatus is an economically important marine bony fish species that is cultured in China on a large scale.However,very little is known about its immune-related genes.In this study,the transcriptome of the immune organs ofP.stellatus that were intraperitoneally challenged with the pathogen Edwardsiella ictaluri JCM1680 is analyzed.Total RNA from four tissues(spleen,kidney,liver,and intestine) was mixed equally and then sequenced on an Illumina HiSeq 2000 platform.Overall,28 465 813 quality reads were generated and assembled into 43 061 unigenes.Similarity searches against public protein sequence databases were used to annotate 28 291 unigenes(65.7%of the total),368 of which were associated with immunoregulation,including 188 related to immunity response.Additionally,the transcript levels of immunity response unigenes annotated as related to tumor necrosis factor(TNF),TNF receptor,chemokine,major histocompatibility complex,and interleukin-6 were investigated in the different tissues of normal and infected P.stellatus by real-time quantitative PCR.The results confirmed that the unigenes identified in the transcriptome database were indeed expressed and up-regulated in infected P.stellatus.To our knowledge,this is the first report of the sequencing and analysis of the transcriptome of P.stellatus.These findings provide insights into the transcriptomics and immunogenetics of bony fish.

Effect of remifentanil on toll-like receptor 4, NF-κB and IL-6 in rabbit myocardial ischemia/reperfusion model

Objective： To investigate whether remifentanil induced cardioprotecting effect is associated with expression of toll-like receptor 4 （TLR4）, nuclear factor rB （NF-r.B） and serum interleukin -6 （IL-6）. Methods： Fifty rabbits were randomly divided into 5 groups （n=10） according to the treatment： sham operation group （group A）, ischemla-reperfusion group （group B）, low-dose remifentanil group （group C）, mediate-dose remifentanil group （group D）, and high-dose remlfentanil group （group E） Myocardial TLR4 mRNA levels, NF-r.B protein expression and serum levels of IL-6 were observed in 120 min after reperfusion. Results： The myocardial expressions of TLR4 mRNA, NF-rd3 protein and IL-6 level in sera of groups B, C, D and E were elevated compared with group A. However, remifentanil significantly reduced the levels of TLR4 mRNA, NF- r.B protein expression and serum IL-6 in groups C, D and E compared with group B. There were remarkable differences between the groups （P〈O.O1）. Conclusion： Intravenous remifentanil has protective effect against rabbit myocardial ischemia/reperfusion injury. This effect may be associated with TLR4, NF-r.B expressions on myocytes and serum level of IL-6 in a dose-dependent manner

THE ROLE OF RECOMBINANT HUMAN FUSION PROTEIN IL－6／IL－2（CH925） IN HEPADNA VIRUS INFECTION TREATMENT

The effects of CH925, a novel immune modulator, on hepadna virus infection was evaluated. Day-old ducklings were inoculated intravenously with LJ-76 DHBV containing serum. Infected ducklings were then treated with the CH925 and the mixture of IL-2 and IL-6 intravenously and the control ducklings received equivalent normal saline (NS). Blood and liver samples were taken and assayed for DHBV DNA and /or DHBsAg. At the completion of the experiment there was a inhibition of viremia with the CH925 and IL-2 + IL-6. Serum DHBV DNA was detected in 6 of 10 ducks in 100 000 unit/kg dosage group, 7 of 10 ducks in 50 000 unit/kg dosage group and 6 of 10 ducks in IL-2 + IL-6 dosage group, compared with 9 of 10 NS control, and it showed a similar result in circulating DHBsAg. When samples of liver DNA were processed for hybridization, a little difference in the DHBV DNA replication was noted between ducks receiving CH925, IL-2 + IL-6 or NS placebo. It is suggested that CH925 might be a potential remedy in HBV infection treatment.

Association Between IL-6 （Interleukin-6） and Iron Status in Rheumatoid Arthritis Patients

The study was performed to determine whether the srum concentrations of IL （interleukin）-6 are elevated in patients with RA （rheumatoid arthritis） and to investigate the relationship between IL-6 levels and iron status in RA patients. 95 serum samples were obtained, 70 of them from patients with RA who had visited the department of Rheumatology at Al-Sadder medical city in Najaf governorate （Iraq） and 25 age and sex-matched healthy controls. The authors assessed the clinical parameters of the disease, including ESR （erythrocyte sedimentation rate）, CRP （C-reactive protein）, and RF （rheumatoid factor）. Serum levels of iron and TIBC （total iron binding capacity） were measured spectrophotometrically, while TS% （transferrin saturation percentage） and transferrin concentration were calculated mathematically. Serum concentrations of IL-6 （interleukin-6） and ferritin were measured using an ELISA （enzyme-linked immunosorbent assay）. The results of serum concentration of IL-6 （interleukin-6） and ferritin were significantly elevated （P 〈 0.0001） in patients with RA compared to those of healthy controls. On the other hand, serum concentrations of iron, TIBC （total iron binding capacity）, TS% （transferrin saturation percentage） and transferrin concentration were significantly decreased in patients with RA compared with those of healthy controls. These findings suggest that anemia is the most frequent observations in patients with RA and mostly associative with increasing level of interleukin-6.

EFFECT OF ACUPUNCTURE ON SERUM INTERLUKIN-6 IN WOMEN WITH POSTMENOPAUSAL OSTEOPOROSIS

: To observe the effect of acupuncture on serum interlukin-6 (IL-6) level in women with post-menopausal osteoporosis. Methods: 59 cases of postmenopausal osteoporosis women were randomly divided into acupunctue group(n=32) and calcium D group(n = 27). In acupuncture group, Zusanli (ST 36), Sanyinjiao (SP 6), Shenshu (BL 23) and Pishu (BL 20) were punctured, 3 times every week, continuously for 6 months. In control group, patients were ordered to take Calcium D, one pill (containing 1500 mg calcium carbonate and VitD3) every morning, continuously for 6 months. Serum IL-6 was detected using radioimmunoassay. Results: After six months' treatment, the result showed that in acupuncture group serum IL-6 calcium level lowered while in control group serum IL-6 content increased. Statistical analysis indicates that there are no significant differences between two groups or between pre-treatment and post-treatment in every single group( P > 0.05). Conclusion: Although no statistical difference was found between two groups, acupuncture could decrease secretion of IL-6 in postmenopausal osteoporosis women to a certain degree, refrain the activity of osteoclast and improve the quality of bone.

Effects of Bradykinin on the Expression of Interleukin-6 in C6 Glioma Cells

OBJECTIVE There is a close correlation between interleukin-6 （IL-6） and malignant proliferation of gliomas. We investigated the effect of bradykinin on the expression of IL-6 in C6 glioma cells. METHODS Semi-quantitive RT-PCR and radioimmunoassay were used to examine the effect of bradykinin on the expression of IL-6 in C6 glioma cells and on the level of IL-6 in the culture medium. RESULTS Using semi-quantitive RT-PCR, the expression of IL-6 mRNA was examined in C6 glioma cells at 0, 5, 10,15, 30, 60 rain following addition of 1μmol/L bradykinin. There was no statistical difference in expression of IL-6 mRNA between the treatment and control groups （P〉0.05） and IL-6 was not detected in the cell culture medium. CONCLUSION Within an hour, IL-6 expression in C6 glioma cells is not induced by bradykinin, suggesting that its clinical application may be useful as a potential therapeutic agent for tumors of the central nervous system .

Effect of 5-FU on modulation of disarrangement of immune-associated cytokines in experimental acute pancreatitis

AIM： To investigate the effects of 5-Fluorouracil （5-FU） on modulation of pro-inflammatory and anti-inflammatory cytokines in acute pancreatitis and the mechanism of it in the treatment of acute pancreatitis. METHODS： Male Sprague Dawley rats were assigned to 3 Groups： Group A, sham operated rats as controls （n = 7）; Group B, acute pancreatitis induced by ductal injection with 5% sodium cholate at a volume of 1.0 mL/kg without any other treatment; Group C, after the pancreatitis was induced as in Group B, the rats were injected intravenously with 5-FU 40 mg/kg. The animals in Groups B and C were killed at 2, 6 and 24 h after operation （n = 7）, and blood samples were taken for measurement of tumor necrosis factor-α （TNF-α）, interleukin-1 （IL-1）, interleukin-6 （IL-6） （by bioassay）, and interleukin-10 （IL-10）, transforming growth factor-β （TGF-β） （by ELISA）. The wet weight of pancreatic tissue, serum amylase levels and white blood cells were also measured. RESULTS： Four rats in Group B and one in Group C died after pancreatitis was induced. Both pro-inflammatory cytokines （TNF-α, IL-1, IL-6） at the 2 and 6 h period and the anti-inflammatory cytokines （IL-10, TGF-β） at 24 h increased significantly （P 〈 0.05） in rats of Group B. After treatment with 5-FU, TNF-α, IL-1, and IL-6 in serum of rats of Group C were inhibited at 2 and 6 h after operation （P 〈 0.05）, and IL-IO, TGF-13 were inhibited at 24 h compared to Group B （P 〈 0.05）. Obvious improvements in the severity of the acute pancreatitis, including the amylase levels, wet weight of pancreatic tissue and neutrophil counts, were also observed after treatment with 5-FU. CONCLUSION： 5-FU is an anti-metabolic and immunosuppressive agent which can minimize the abnormal immune o/tokine response and relieve the pathophysiological disorders associated with experimental acute pancreatitis.

Inhalation of hydrogen gas reduces liver injury during major hepatotectomy in swine

AIM： To study the effect of H2 gas on liver injury in massive hepatectomy using the Intermittent Pringle maneuver in swine. METHODS： Male Bama pigs （n = 14） treated with ketamine hydrochloride and Sumianxin Ⅱ as induc- tion drugs followed by inhalation anesthesia with 2% isoflurane, underwent 70% hepatotectomy with loss of bleeding less than 50 mL, and with hepatic pedicle occlusion for 20 min, were divided into two groups： Hydrogen-group （n = 7）, the pigs with inhalation of 2% hydrogen by the tracheal intubation during major hepa- totectomy; Contrast-group （n= 7）, underwent 70% hepatotectomy without inhalation of hydrogen. Hemo- dynamic changes and plasma concentrations of alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, hyaluronic acid （HA）, tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6）, and malondialdehyde （MDA） in liver tissue were measured at pre-operation, post-hepatotectomy （PH） 1 h and 3 h. The apoptosis and proliferating cell nuclear antigen （PCNA） expres- sion in liver remnant were evaluated at PH 3 h. Then we compared the two groups by these marks to evalu- ate the effect of the hydrogen in the liver injury during major hepatotectomy with the Pringle Maneuver in the swine. RESULTS： There were no significant differences in body weight, blood loss and removal liver weight be- tween the two groups. There was no significant differ- ence in changes of portal vein pressure between two groups at pre-operation, PH 30 min, but in hydrogen gas treated-group it slightly decrease and lower than its in Contrast-group at PH 3 h, although there were no significant difference （P = 0.655）. ALT and AST in Hydrogen-group was significantly lower comparing to Contrast-group （P = 0.036, P = 0.011, vs P = 0.032, P = 0.013） at PH 1 h and 3 h, although the two groups all increased. The MDA level increased between the two group at PH i h and 3 h. In the hydrogen gas treated- group, the MDA level was not significantly significant at pre-operation and significantly low at PH 1 h and 3 h comparing to Contrast-group （P = 0.0005, P = 0.0004）. In Hydrogen-group, the HA level was also significantly low to Contrast-group （P = 0.0005, P = 0.0005） al- though the two groups all increased at PH 1 h and 3 h. The expression of cluster of differentiation molecule 31 molecules Hydrogen-group was low to Contrast-group. However, PCNA index （%） was not statistically signifi- cant between the two groups （P = 0.802）. Micropho- tometric evaluation of apoptotic index （AI） in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-stained tissue after hepatotectomy for 3h, the AI% level in the hydrogen was significantly low to Contrast-group （P = 0.012）. There were no significant difference between Hydrogen-group and Contrast- group at pre-operation （P = 0.653, P = 0.423）, but after massive hepatotectomy, the TNF-α and IL-6 levels increase, and its in Hydrogen-group was significantly low compared with Contrast-group （P = 0.022, P = 0.013, vs P = 0.016, P= 0.012）, respectively. Hydro- gen-gas inhalation reduce levels of these markers and relieved morphological liver injury and apoptosis.CONCLUSION： H2 gas attenuates markedly ischemia and portal hyperperfusion injury in pigs with massive hepatotectomy, possibly by the reduction of inflamma- tion and oxidative stress, maybe a potential agent for treatment in clinic.

Serum inter-cellular adhesion molecule 1 is an early marker of diagnosis and prediction of severe acute pancreatitis

AIM:To determine if serum inter-cellular adhesion molecule 1(ICAM-1)is an early marker of the diagnosis and prediction of severe acute pancreatitis(SAP) within 24 h of onset of pain,and to compare the sensitivity,specificity and prognostic value of this test with those of acute physiology and chronic health evaluation(APACHE)Ⅱscore and interleukin-6(IL-6). METHODS:Patients with acute pancreatitis(AP)were divided into two groups according to the Ranson's criteria:mild acute pancreatitis(MAP)group and SAP group.Serum ICAM-1,APACHEⅡand IL-6 levels were detected in all the patients.The sensitivity,specificity and prognostic value of the ICAM-1,APACHEⅡscore and IL-6 were evaluated. RESULTS:The ICAM-1 level in 36 patients with SAP within 24 h of onset of pain was increased and was significantly higher than that in the 50 patients with MAP and the 15 healthy volunteers(P<0.01).The ICAM-1 level(25 ng/mL)was chosen as the optimum cutoff to distinguish SAP from MAP,and the sensitivity,specificity,positive predictive value,negative predictive value(NPV),positive likelihood ratio and negative likelihood ratio were 61.11%,71.42%,0.6111,0.7142, 2.1382 and 0.5445,respectively.The area under the curve demonstrated that the prognostic accuracy of ICAM-1(0.712)was similar to the APACHE-Ⅱscoring system(0.770)and superior to IL-6(0.508)in distinguishing SAP from MAP. CONCLUSION:ICAM-1 test is a simple,rapid and reliable method in clinical practice.It is an early marker of diagnosis and prediction of SAP within the first 24 h after onset of pain or on admission.As it has a relatively low NPV and does not allow it to be a stand-alone test for the diagnosis of AP,other conventional diagnostic tests are required.

Side-stream smoking reduces intestinal inflammation and increases expression of tight junction proteins

AIM:To investigate the effect of side-stream smoking on gut microflora composition,intestinal inflammation and expression of tight junction proteins.METHODS:C57BL/6 mice were exposed to side-stream cigarette smoking for one hour daily over eight weeks.Cecal contents were collected for microbial composition analysis.Large intestine was collected for immunoblotting and quantitative reverse transcriptase polymerase chain reaction analyses of the inflammatory pathway and tight junction proteins.RESULTS:Side-stream smoking induced significant changes in the gut microbiota with increased mouse intestinal bacteria,Clostridium but decreased Fermicutes(Lactoccoci and Ruminococcus),Enterobacteriaceae family and Segmented filamentous baceteria compared to the control mice.Meanwhile,side-stream smoking inhibited the nuclear factor-κB pathway with reduced phosphorylation of p65 and IκBα,accompanied with unchanged mRNA expression of tumor necrosis factor-α or interleukin-6.The contents of tight junction proteins,claudin3 and ZO2 were up-regulated in the large intestine of mice exposed side-stream smoking.In addition,side-stream smoking increased c-Jun N-terminal kinase and p38 MAPK kinase signaling,while inhibiting AMPactivated protein kinase in the large intestine.CONCLUSION:Side-stream smoking altered gut microflora composition and reduced the inflammatory response,which was associated with increased expression of tight junction proteins.

Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophages

Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40μmol/ L) inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0. 5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.

Effect of dexamethasone,anisodamine and rhubarb therapy on rats with acute pancreatitis

Objective:To investigate the therapeutic effects of dexamethasone,anisodamine and rhubarb(DAR) on endotoxin,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and pancreatic damage in rat models of acute pancreatitis(AP).Methods:The AP rat models were prepared and randomly assigned to AP group(n=10) and DAR group(n=10),while other healthy rats were assigned to the sham-operated group(n=10).The rats were euthanized at 6 h after operation,and then the serum levels of endotoxin,TNF-α,IL-6 and histology of pancreas were determined as the indexes of therapeutic effects.Results:At 6 h after operation,serum levels of endotoxin,TNF-α and IL-6,and pancreatic damage were significantly increased in AP group compared with those in sham-operated group(P<0.01).Compared with the AP group,DAR therapy remarkably attenuated the endotoxin,TNF-α,IL-6 levels and reduced pancreatic damage(P<0.05).Conclusion:The inhibition of pancreatic damage by DAR in rats with AP might contribute,in part at least,to the amelioration of pancreatic inflammation.The present study provides beneficial evidence that DAR may be useful in the treatment of AP model of rats.