Cloning,Identification and Differential Expression Analysis of Full-length cDNA of Carp Interleukin-1β

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of carp interleukin-1β （IL-1β） cDNA. [Method] By using DD-RTPCR method,the differential expression cDNA fragments were gained. The cDNA library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the full length cDNA of carp IL-1β was cloned. Moreover,the sequence analysis and differential expression analysis were carried out. [Result] The positive clone which had a whole ORF that encoded 276 amino acids was obtained. The cluster analysis showed that the amino acid sequence of carp IL-1β and Japanese carp closely gathered as a branch,and the homoeology of amino acid sequence reached 95%. The clustering order was the carassius,zebra fish,pig,cattle,horse,human and mouse in turn. The differential expression analysis showed that the expression of IL-1β in the leucocytes significantly increased in the prior period （4 h） after the mitogen stimulated. But as the time went by （12 and 24 h）,it didn＇t increase in the same period. The total trend of expression amount presented the peak type. [Conclusion] The research laid the foundation for further studying the expression manner,function characteristic,regulation mechanism of IL-1β in vivo and its action mechanisms in the inflammatory reaction,emergency reaction and immune response.

Changes of gastric and intestinal blood flow, serum phospholipase A_2 and interleukin-1β in rats with acute necrotizing pancreatitis

AIM:To explore the relationship between gastric and intestinal microcirculatory impairment and inflammatory mediators released in rats with acute necrotizing pancreatitis (ANP). METHODS: A total of 64 rats were randomized into control group and ANP group. ANP model was induced by injection of 5% sodium taurocholate under the pancreatic membrane. Radioactive biomicrosphere technique was used to measure the gastric and intestinal tissue blood flow at 2 and 12 h after the induction of ANP, meanwhile serum phospholipase A2 (PLA2) activities and interleukin-1β levels were determined. Pathologic changes in pancreas, gastric and intestinal mucosae were studied. RESULTS: The gastric blood flow in ANP group (0.62±0.06 and 0.35±0.05) mL/(min·g) was significantly lower than that in control group (0.86±0.11 and 0.85±0.06) mL/(min·g) (P<0.01) at 2 and 12 h after induction of ANP. The intestinal blood flow in ANP group (0.80±0.07 and 0.50±0.06) mlV(min·g) was significantly lower than that in control group (1.56±0.18 and 1.61±0.11) mL/(min·g) (P<0.01). Serum PLA2 activities (94.29±9.96 and 103.71± 14.40) U/L and IL-1β levels (0.78±0.13 and 0.83±0.20)μg/L in ANP group were higher than those in control group (65.27±10.52 and 66.63±9.81) U/L, (0.32±0.06 and 0.33±0.07)μg/L (P<0.01). At 2 and 12 h after introduction of the model, typical pathologic changes were found in ANP. Compared with control group, the gastric and intestinal mucosal pathologic changes were aggravated significantly (P<0.01) at 12 h after induction of ANP. Gastric and intestinal mucosal necrosis, multiple ulcer and hemorrhage occurred. CONCLUSION: Decrease of gastric and intestinal blood flow and increase of inflammatory mediators occur simultaneously early in ANP, both of them are important pathogenic factors for gastric and intestinal mucosal injury in ANP.

Interleukin-1β gene polymorphism associated with hepatocellular carcinoma in hepatitis B virus infection

AIM： To examine the effect of interleukin-l-beta （IL-113） promoter region C-511T and IL-1 receptor antagonist （IL-1RN） polymorphism among the patients with chronic hepatitis B virus （HBV） infection （HCC and non-HCC）. METHODS： Genomic DNA from 136 Thai patients with chronic HBV infection （HCC =46 and non-HCC= 90） and 152 healthy individuals was genotyped for IL-113 gene polymorphism （-511） using polymerase chain reaction with sequence specific primers （PCR-SSP）. The variable number of tandem repeats （VNTR） of IL-1RN gene was assessed by a PCR-based assay. The association between these genes and status of the disease was evaluated by X^2 test. RESULTS： IL-1B-511 genotype c/c was found to be significantly different in patients with HCC when compared with healthy individuals （P = 0.036, OR = 2.29, 95%CI = 1.05-4.97） and patients without HCC （P=0.036, OR= 2.52, 95%CI=1.05-6.04）. Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in healthy control （P=0.033, OR= 1.72, 95%CI=1.04-2.84）. However, no significant association in IL-1RN gene was found between the two groups. CONCLUSION： IL-1B-511C allele, which may be associated with high IL-1B production in the liver, is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.

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.

细胞焦亡在阿片类药物所致痛觉过敏中的作用

阿片类药物通常为手术镇痛的首选用药,但其使用往往会引发阿片类药物所致痛觉过敏(opioidinduced hyperalgesia,OIH)等不良反应。以往研究证实,OIH的发生可能与大脑中枢激活相关。研究发现,脊髓胶质细胞活化后炎性介质释放可介导OIH发生,而含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteinase1,caspase1)介导的细胞焦亡通路作为释放炎性介质的重要途径之一,可能参与了小胶质细胞的炎性反应。文章基于目前研究成果,对细胞焦亡通路特性、OIH的发生机制等方面进行综述,从脊髓胶质细胞活化、细胞焦亡通路介导其他类型OIH和炎性介质释放等方面来探讨细胞焦亡通路和OIH之间的可能关系,为OIH的治疗提供新思路。

Effect of Moxibustion on IL-1β and IL-2 in Rat Models of Rheumatoid Arthritis

Objective： To observe the influence on IL-1β and IL-2 in rat models with rheumatoid arthritis after moxibustion on Shenshu （BL 23） and Zusanli （ST 36） points, and to discuss the mechanism of moxibustion. Methods： Fifty male Wistar rats were divided randomly into 5 groups, control group, model group, drug group, moxibustion group, and laser group, 10 for each. Four groups except the normal group were built on the model of rheumatoid arthritis. The changes of body weight and plantar circumference were measured and the level of IL-1β, IL-2 in sera were examined by ELISA. Results： Compared with the model group, the weight and plantar circumference of rats in the moxibustion group were improved significantly after treatment （P〈0.01）, and the improvement of plantar circumference also had significant differences compared with the drug group and the laser group （P〈0.05）. The level of IL-1β, IL-2 in sera were down regulated in the moxibustion group and the laser group, which had statistical differences compared with the model group （P〈0.05）, but no statistical differences were found when comparing with the drug group. Conclusion： Moxibustion obviously improves the toe tumefaction of the rats with rheumatoid arthritis, which is better than CO2 laser of 10.6μm. On the aspect of decreasing the amount of IL-1β, IL-2, CO2 laser of 10.6μm is similar with moxibustion.

pORF5 plasmid protein of Chlamydia trachomatis induces MAPK-mediated pro-inflammatory cytokines via TLR2 activation in THP-1 cells

Infection with Chlamydia trachomatis induces inflammatory pathologies in the urogenital tract that can lead to infertility and ectopic pregnancy. Pathogenesis of infection has been mostly attributed to excessive cytokine production. However, precise mechanisms on how C. trachomatis triggers this production, and which protein（s） stimulate inflammatory cytokines remains unknown. In the present study, the C. trachomatis pORF5 protein induced tumor necrosis factor alpha （TNF-a）, interleukin-1 beta （IL-1β） and interleukin-8 （IL-8） in dose and time-dependent manners in the THP-1 human monocyte cell line. We found that intracellular p38/mitogen-activated protein kinase （MAPK） and extracellular signal-regulated kinase （ERK）/MAPK signaling pathways were required for the induction of TNF- a, IL-1β and IL-8. Blockade of toll-like receptor 2 （TLR2） signaling reduced induction levels of TNF-a, IL-8 and IL-1β. We concluded that the C. trachomatis pORF5 protein might contribute to the inflammatory processes associated with chlamydial infections.

Changes of interleukin-1β, tumor necrosis factor α and interleukin-6 in brain and plasma after brain injury in rats

Objective: To study the changes of interleukin 1 β (IL 1β), tumor necrosis factor α (TNFα) and interleukin 6 (IL 6) levels in brain and plasma after brain injury and to assess the relationship between the cytokine levels and injury severity in rats. Methods: A total of 51 male Wistar rats, weighing 280 340 g , were anesthetized with chloral hydrate ( 400 mg/kg body weight) through intraperitoneal injection and fixed on a stereotaxic instrument. Severe brain injury was created in 16 rats (severe injury group) and moderate brain injury in 18 rats (moderate injury group) by a fluid percussion model, and cytokine levels of IL 1β, TNFα and IL 6 were measured with biological assay. And sham operation was made on the other 17 rats (control group). Results: In the control group, the levels of IL 1β, TNFα and IL 6 were hardly detected in the cortex of the rats, but in the ipsilateral cortex of the rats in both injury groups, they increased obviously at 8 hours after injury. The increasing degree of these cytokines had no significant difference between the two injury groups. The levels of IL 6 in the plasma of all the rats increased slightly, whereas the levels of IL 1β and TNFα were undetectable. Conclusions: The increase of IL 1β, TNFα and IL 6 levels is closely related to brain injury. The increased cytokine levels in the central nervous system are not parallel to those in the peripheral blood. It suggests that inflammatory cytokines play important roles in the secondary neural damage after brain injury.

Effect of moxibustion plus Duhuo Jisheng decoction on hs-CRP,IL-1β and TNF-α levels in middle-aged and elderly patients with knee osteoarthritis

Objective： To observe the clinical effect of moxibustion combined with Duhuo Jisheng decoction for middle-aged and elderly patients with knee osteoarthritis（KOA） and its impact on serum high sensitive C-reactive protein（hs-CRP）,interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α） levels.Methods： A total of 90 eligible KOA patients were randomized into an observation group（n=45） and a control group（n=45）.Cases in the observation group received moxibustion plus oral administration Duhuo Jisheng decoction, while cases in the control group received Duhuo Jisheng decoction. Then the hs-CRP, IL-1β and TNF-α levels were examined and the clinical effect was evaluated.Results： Before treatment, the hs-CRP, IL-1β and TNF-α levels showed no between-group statistical differences（all P〉0.05）. After 8 weeks of treatment, the hs-CRP, IL-1β and TNF-α levels all dropped significantly in both groups（P〈0.01 or P〈0.05）, and the treatment group showed more substantial changes than the control group（all P〈0.05）. The total effective rate was 97.8% in the treatment group, versus 86.7% in the control group, showing a statistical difference（P〈0.05）.Conclusion： Moxibustion plus Duhuo Jisheng decoction has a good clinical effect for middle-aged and elderly KOA patients,and the effect may relate to the decreased hs-CRP, IL-1β and TNF-α levels.

Effect of modified Qing Long Bai Wei needling on the levels of IL-1β, IL-6 and INF-α in synovial fluid of knee osteoarthritis patients

Objective:To compare the modified Qjng Long Bai Wei needling method and ordinary acupuncture method in the effects of improving the levels of interleukin (IL)-1β,IL-6 and tumor necrosis factor (TNF)-α in the treatment of knee osteoarthritis (KOA),and to determine the advantage of the modified Qing Long Bai Wei needling method for KOA.Methods:One hundred KOA patients were randomized into a treatment group and a control group by using the random number table,with 50 cases in each group.The treatment group was intervened by the modified Qing Long Bai Wei needling method,and the control group was given ordinary acupuncture.The two groups were observed before and after the treatment to determine the changes in the levels of IL-1β,IL-6 and TNF-α in synovial fluid,and the clinical efficacies were compared between the two groups.Results:The total effective rate and clinical recovery rate were 97.9％ and 52.1％ respectively in the treatment group,versus 85.1％ and 25.5％ in the control group,and the between-group differences were statistically significant (both P＜0.01).After the treatment,the levels of Ib1β,IL-6 and TNF-cα in synovial fluid changed significantly in both groups (all P＜0.01);there were significant differences in the levels of IL-1β,IL-6 and TNF-α in synovial fluid between the two groups (all P＜0.01).Conclusion:The modified Qjng Long Bai Wei needling is an effective method for KOA and it can significantly improve the levels of IL-1β,IL-6 and TNF-α in synovial fluid.

The mechanism of galanthamine regulating IL-1β/IL-1RA ratio to ameliorate inflammatory microenvironment

In the present study,we aimed to evaluate the anti-inflammatory mechanism of galanthamine,a classic therapeutic drug for Alzheimer s disease(AD).The co-culture system of hippocampal nerve cell line HT-22 and microglial cell line BV-2 was established to observe the effect of galanthamine on the expressions of inflammatory factors induced by lipopolysaccharide(LP S).MTT method was used to observe the protective effect of galanthamine on neurons.ELISA and qPCR methods were used to detect the expressions of interleukin-β(IL-1β) and IL-1 receptor antagonist(IL-1 RA) at the protein and mRNA levels,respectively.IL-1β and IL-1 RA were evaluated by the ELISA method after pretreating with galanthamine and α7 nAChR blockerα-bungarotoxin(α-bun),mAChR blocker atropine(Atr),PI3 K inhibitor LY294002,IKKβ inhibitor SC514,or MEK inhibitor PD98059,respectively.The results showed that galanthamine significantly inhibited LPS-induced increased IL-1β and IL-1 RA expressions and maintained the ratio of IL-1β/IL-1 RA.α-Bun could block the regulatory effect of galanthamine on IL-1β and IL-1 RA.As PI3 K and NF-κB pathways were blocked,the regulatory effect of galanthamine on the IL-1β expression was significantly inhibited.Blocking PI3 K and MEK pathways could significantly inhibit the regulation of galanthamine on IL-1 RA expression.In summary,galanthamine regulated the inflammatory activity of the IL-1 subfamily to play an anti-inflammatory role mediated by α7 nAChR and PI3 K/NF-κB/MAPK pathways,which probably provided a new strategy for AD treatment.

Analysis of polymorphisms in the promoter region of interleukin-1 β by restriction fragment length polymorphism-PCR

Objective: To investigate the frequencies of -1470, (-511) and -31 single nucleotide polymorphisms (SNPs) in the promoter of IL-1β and its haplotype constitution in Chongqing population. Methods: One hundred and twelve healthy Chongqing people were enrolled in this study. Polymorphisms at -1470 (G to C), (-511) (T to C) and -31 (C to T) of IL-1β were genotyped with the method of restriction fragment length polymorphism (RFLP). Haplotype frequencies were analyzed by Arlequine software.Results: Frequencies of IL-1β -1470, -511 and -31 SNPs were (41.67)%, 50% and (45.33)%, respectively. Genotype frequencies of -1470 locus were (39.81)%, (37.04)% and (23.15)% for G/G, G/C and C/C respectively. As for T-511C SNP, genotype frequencies of (T/T), T/C and C/C were (29.91)%, (40.18)% and (29.91)%, respectively. Genotyping results of C/C, C/T, and T/T of -31 locus were (35.51)%, (38.32)% and (26.71)% respectively. Haplotype analysis found that there were mainly three haplotypes constituted by three SNPs, ie., G-T-C, C-T-C and G-C-T. Conclusions: Polymorphisms exist in the promoter of IL-1β in Chongqing population. Three SNPs locate in the same haplotype block.

Regulatory effects of moxibustion on ubiquitin and NLRP3 proteins in colon of ulcerative colitis rats

Objective:To observe the effects of moxibustion on colonic inflammation,and the expressions of ubiquitin and nucleotide-binding oligomerization domain(Nod)-Iike receptor protein 3(NLRP3)proteins in rats with ulcerative colitis(UC),and to explore the anti-inflammatory mechanism of moxibustion in the UC treatment.Methods:Clean grade male Sprague-Dawley(SD)rats were randomly divided into a normal group(NG),a model group(MG),a moxa-stick moxibustion group(MSMG)and a Western medicine group(WMG).UC model was prepared by freely drinking 35 g/L d ext ran sulfate sodium(DSS)solution.Bilateral Tianshu(ST 25)were selected for mild moxibustion treatment in the MSMG;mesalazine solution was intragastrically administrated in the WMG.Rats in the NG and MG were only grasped and fixed as in the MSMG without any treatment.After treatment,hematoxylin-eosin(HE)staining was performed to observe and score the colonic pathological damage under light microscope;immunofluorescence method was used to determine the expression of colonic ubiquitin protein;immunohistochemical method was used to detect the expressions of colonic interleukin(IL)-1β and NLRP3 proteins.Results:The colon tissue was severely injured,and the pathological score was significantly increased in the MG than in the NG(P<0.01),and the protein expressions of ubiquitin,NLRP3 and IL-1β in the colon were significantly increased(all P<0.01).Compared with the MG,the colonic damage was repaired,the inflammation and pathological scores were reduced,and the ubiquitin,NLRP3 and IL-1β protein expressions were decreased in the MSMG and WMG(all P<0.01).Correlation analysis revealed that the ubiquitin protein expression was correlated with the colonic pathological score and the NLRP3 protein expression(r=0.677,P<0.01;r=0.536,P<0.05).Conclusion:Moxibustion can down-regulate the protein expressions of ubiquitin,NLRP3 and IL-1β in the colon of UC rats,which may be one of the mechanisms to promote the repair of colonic inflammatory lesions and exert anti-inflammatory effects.