| Resistin was initially studied as a signal molecule to promote insulin resistance,but now it is more considered to be an effector molecule of proinflammatory or inflammatory factors,which participates in the development of inflammation.Resistin activates porcine alveolar macrophages(PAMs)and promotes the expression of IL-1β,IL-6 and TNF-α,and TLR4 is an important signaling pathway involved in inflammation and immunity.In order to further clarify the mechanism of action between resistin and bovine alveolar macrophages(BAMs),the recombinant bovine resistin was expressed in prokaryotic cells,then investigated the effects on Blood Routine Indexes of mice and the expression and regulation of inflammatory cytokines in BAMs.1.Bovine resistin cloning and its high level of soluble expression.Total RNA was extracted from the omental adipose tissue of healthy calves,amplified primers were designed according to the sequence of bovine resistin gene in Genbank(NM-183362.1).The target gene was amplified by RT-PCR,then sequenced by PMD19-T vector and cloned into the prokaryotic expression vector PET-21 a and constructed recombinant expression plasmid PET-Resistin.The expression conditions of recombinant resistin include host bacteria(E.coli BL21 and E.coli Rosetta),IPTG concentration(0,0.1,0.2,0.4,0.6,0.8 and 1.0 mmol/L),induction temperature(20℃,25℃,30℃ and 35℃)and time(10 h,12 h,14 h and 16 h)were optimized.The fusion protein was identified by anti-His label antibody and purified by nickel column.The results showed that when the final concentration of IPTG was 1.0 mmol/L and the induction time was 14 h at 20 ℃,the host strain Rosetta expressed the highest amount of bovine resistin.The recombinant resistin existed in the supernatant and could be purified by nickel column filtration.2.Effects of recombinant bovine resistin on blood routine parameters.One hundred-twenty healthy Kunming mice were randomly divided into four groups,30 mice in each group.Groups I,II,III and IV were intraperitoneally injected with recombinant resistin at doses of 0 mg/mice,0.25 mg/mice,0.5 mg/mice and 1.0 mg/mice,respectively.Blood samples were collected at 0 h(before injection),6 h and 24 h afterinjection.Heparin anticoagulation was used to detect blood routine parameters.The results showed that:(1)WBC,at 6 h,groups II,III and IV were significantly higher than group I,group III was significantly higher than groups II and IV(P<0.01);at 24 h,groups II and III were significantly higher than groups I and IV,and group II was significantly higher than group III(P<0.01).(2)Neu,at 6 h,groups II,III and IV were significantly higher than group I,groups III and IV were significantly higher than group II(P<0.01);at 24 h,group III was significantly higher than groups I and IV(P<0.01).(3)Lym,at 6 h,groups III and IV were significantly higher than groups II and I(P<0.01);at 24 h,groups II and III were significantly higher than groups I and IV,group II was significantly higher than group III(P<0.01).(4)At 24 h,Mon and EOS of group II were significantly higher than groups I,III and IV(P<0.01).(5)Bas,24 h,group II was significantly higher than groups I,III and IV,and group III and IV were significantly higher than group I(P<0.01).(6)At 6 h and 24 h,RBC,HCT and HGB of groups II,III and IV were significantly lower than group I(P<0.01).(7)PLT,at 6 h,group IV was significantly lower than groups I,II and III(P<0.01);at 24 h,groups II,III,IV were significantly lower than group I(P<0.01 or P<0.05),group II was significantly lower than group III(P<0.01).(8)PCT,at 6 h,groups III and IV were significantly lower than groups I and II(P<0.01 or P<0.05),group III group was significantly higher than group IV(P < 0.01);at 24 h,groups II,III and IV were significantly lower than group I(P<0.01).The above results showed that recombinant resistin had biological activity and could promote the increase of white blood cells(mainly Neu and Lym)in the blood of mice,and reduce the effects of RBC,HGB and PLT.3.Effects of recombinant bovine resistin on release of inflammatory cytokines and expression of related genes in bovine alveolar macrophages.The experiment was divided into groups I,II,III and IV,BAMs were incubated with recombinant resistin at final concentrations of 0 ng/mL,10 ng/mL,100 ng/mL and 500ng/mL,respectively.Cell supernatants and lower layers were collected and cultured at 0 h,1.5 h,3 h,6 h,12 h and 24 h.The concentrations of IL-1β、IL-6 and TNF-α in supernatant and the changes of gene and protein expression of PPARγ,TLR4,MyD88,IRAK4,TRAM and NF-κB in lower layer cells were detected.Result:(1)Changes of inflammatory cytokines in supernatant:(1)IL-1β,groups III and IV were significantly higher than groupsI and II at 1.5 h,3 h,6 h,12 h,24 h,of which group III was significantly higher than group I at 3 h,6 h,12 h and 24 h(P<0.05);group IV was significantly higher than group III at1.5 h,3 h and 6 h,and significantly lower than group III at 24 h(P<0.05).(2)IL-6,groups II,III and IV were significantly higher than group I at 1.5 h,3 h,6 h,12 h,24 h,in which groups III and IV were significantly higher than group II(P < 0.05);group IV was significantly higher than group III at 1.5 h,3 h,6 h and 12 h,and significantly lower than group III at 24 h(P<0.05).(3)TNF-a,groups III and IV were significantly higher than groups I and II at 1.5 h,3 h,6 h,12 h,in which group IV was significantly higher than group III at 1.5 h,3 h and 6 h,and significantly lower than group III at 12 h and 24 h(P<0.05);group II was significantly higher than group I at 3 h,6 h,12 h and 24 h,groups III and IV were significantly higher than group II at 24 h(P<0.05).These results indicate that recombinant resistin can promote the release of pro-inflammatory cytokines such as IL-1β、IL-6 and TNF-α,and the effect is related to the concentration and time of action of recombinant resistin.(2)Changes of BAMs mRNA: PPARγ,groups III and IV at 1.5 h,3 h,6 h,12 h,24 h and group II at 6 h,12 h,24 h were significantly lower than group I(P <0.05);group II was significantly higher than groups III and IV at 1.5 h and 3 h,and significantly lower than group IV at 12 h and 24 h(P < 0.05);group IV was significantly higher than group III at 12 h and 24 h(P < 0.05).TLR4,MyD88,IRAK4,TRAM,NF-κB,groups II,III and IV were significantly higher than group I at 6,12,24 h,and group II,III and IV of TRAM was also significantly higher than group I at 3 h(P < 0.05);TLR4,groups III and IV were significantly higher than that group II at 6,12,24 h,group IV was significantly higher than group III at 12 h(P < 0.05);MyD88,groups III and IV were significantly higher than group II at 6,12 h(P < 0.05);IRAK4,groups III and IV were significantly higher than that group II at 6,12,24 h,groups III was significantly higher than groups IV at 12,24 h(P < 0.05);TRAM,groups III and IV were significantly higher than group II at 6,12 h,and significantly lower than group II at 24 h,group IV was significantly lower than group III at 12 h and significantly higher than groups III at 24 h(P< 0.05);NF-κB,groups III and IV were significantly higher than group II at 6 h and 12 h,group IV was significantly higher than groups II and III at 24 h(P < 0.05).The results showed that recombinant resistin could up-regulate the expression of TLR4,MyD88,IRAK4,TRAM and NF-κB genes in BAMs,and inhibit the expression of PPARγ gene,but the inhibition of PPARγ gene expression was prior to that of other genes.(3)Protein changes of BAMs: the expression of PPARγ protein in group III at 12 h was significantly lower than group I(P < 0.05),while the expression of TLR4,MyD88,IRAK4,TRAM and NF-κB was significantly higher than group I(P < 0.05),which was consistent with the result of the expression of mRNA.The results showed that recombinant resistin can inhibit the expression of PPARγ in BAMs,and then up-regulate the expression of TLR4,MyD88,IRAK4,TRAM and NF-κB.In conclusion1.A highly efficient and soluble expression system of recombinant bovine resistin was successfully constructed: host strain Rosetta,the final concentration of IPTG was 1.0mmol/L,induction temperature was 20℃,induction time was 14 h,and which was purified by nickel column filtration2.Recombinant bovine resistin has biological activity.Intraperitoneal injection of recombinant bovine resistin can promote the increase of white blood cells(mainly Neu and Lym)in mice blood,and reduce RBC,HGB and PLT.3.Recombinant resistin can activate TLR4/NF-κB signaling pathway and promote the release of pro-inflammatory cytokines such as IL-1β,IL-6 and TNF-α in BAMs.The mechanism is related to the inhibition of PPARγ by recombinant resistin and the up-regulation of the expression of TLR4,MyD88,IRAK4,TRAM and NF-κB genes and proteins. |
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