| Sepsis is a state of pathological and physiological abnormal reactions caused by infection. As the main reasons of death and disability in patients with infection worldwide, sepsis remains a focal point in medical field. Our previous studies demonstrated that combined administration of 0.5 minumum alveolar concentration(MAC) isoflurane with 60% oxygen can protect against experimental sepsis. In addition, using mi RNA microarray assay followed by quantitative real-time PCR, we got 4 sepsis-associated mi RNAs--- mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p and mi R-542-3p. But it is unclear how these 4 mi RNAs function in neutrophil, monocyte as well lymphocyte. It has been acknowledged that macrophage is critical in the pathological and physiological process of sepsis. Our preliminary studies showed that LPS stimulated significantly increased expression of mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p and mi R-542-3p in RAW264.7 cells. So, this project was scheduled to study the roles of mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p and mi R-542-3p in the protective actions against sepsis by 0.5MAC isoflurane and 60% oxygen, and to explore whether these 4 mi RNAs became potential targets for developing newer measures for treatment of sepsis.Materials and Methods1. 4 mi RNAs contributed to the protective action against sepsis provided by the 0.5 MAC isoflurane in 60% oxygen through NF-?B pathways(1) In-vitro study: RAW264.7 cell lines were used for this study. Cells were randomly assigned to the following 10 groups: Vehicle, LPS, Vehicle+0.5MAC ISO+60% Oxy, LPS+0.5MAC ISO+60% Oxy,LPS+LV-/mimic-/antigomo-mi R- 133a-3p+0.5MAC ISO+60% Oxy, LPS+LV-/mimic-/antigomo-mi R- 141-3p+0.5MAC ISO+60% Oxy, LPS+LV-/mimic-/antigomo-mi R-3074-2-3p+0.5MAC ISO+60% Oxy, LPS+LV-/mimic-/antigomo-mi R-542-3p+0.5MAC ISO+60% Oxy, LPS+LV-/mimic-/ antigomo-mi R-mix+0.5MAC ISO+60% Oxy and LPS+LV-/mimic-/antigomo- NC+ 0.5MAC ISO+60% Oxy groups. An in-vitro sepsis model was induced by 100 ng/ml LPS stimulation for at least 2 h. Lentivirus carrying LV3-mmu-mi R-133a-3p, LV3-mmu-mi R-141-3p, LV3-mmu-mi R-3074-2-3p or LV3-mmu-mi R-542-3p expression plasmid were infected to RAW264.7 cells 72 h before LPS stimulation, and mimics or antigomos for the above 4 mi RNAs were transfected to the cells 12 h before LPS stimulation. Treatment: cells were exposured to 0.5MAC isoflurane in 60% oxygen for 2 h at the beginning of LPS stimulation. Cell culture supernatants were harvested for determination of inflammatory cytokines TNF-? and IL-1?. Proteins were extracted for assay of p-p65 and p65 expressions in cytoplasm and total cells, and p65 expression in nuclei using western blot technique, respectively.(2) Animal study: Male Kunming mice(ICR/Km) were randomly assigned to the following 9 groups: Sham+Air, CLP+Air, CLP+0.5MAC ISO+60% Oxy, CLP+LV-NC+0.5MAC ISO+60% Oxy, CLP+LV-mi R-133a-3p+0.5MAC ISO+60% Oxy, CLP+LV-mi R-141-3p+0.5MAC ISO+60% Oxy, CLP+LV-mi R-3074-2-3p+ 0.5MAC ISO+60% Oxy, CLP+LV-mi R-542-3p+0.5MAC ISO+60% Oxy and CLP+ LV-mi R-mix+0.5MAC ISO+60% Oxy groups. CLP was performed on ICR/Km mice for induction of sepsis. 48 h before CLP procedure, lentivirus infection in animals were conducted by injection of lentivirus carrying LV3-mmu-mi R-133a-3p, LV3-mmu-mi R-141-3p, LV3-mmu-mi R-3074-2-3p or LV3-mmu-mi R-542-3p expression plasmid via tail vein. Treatment: animals were exposed to 0.5MAC isoflurane in 60% oxygen for 1 hour starting at 1 and 6 h after CLP/Sham-operation, respectively. Survival rate was observed for 7 days.2. Inhibiton to mi RNAs proteced against sepsis(1) Animal study: Male ICR/Km were randomly assigned to the following 8 groups: Sham, CLP, CLP+LV-NC inhibitor/antigomo-NC, CLP+LV-mi R-133a-3p inhibitor/ antigomo-mi R-133a-3p, CLP+LV-mi R-141-3p inhibitor/antigomo-mi R-141-3p, CLP+ LV-mi R-3074-2-3p inhibitor/antigomo-mi R-3074-2-3p, CLP+LV-mi R-542-3p inhibitor/antigomo-mi R-542-3p and CLP+LV-mi R-mix inhibitor/antigomo-mi R-mix groups. CLP was performed on ICR/Km mice for induction of sepsis. 48 h before CLP procedure, lentivirus infection in animals were conducted by injection of lentivirus containing the LV3-mmu-mi R-133a-3p-inhibitor, LV3-mmu-mi R-141-3p-inhibitor, LV3-mmu-mi R-3074-2-3p-inhibitor,or LV3-mmu-542-3p-inhibitor expression plasmid via tail vein. Transfection of antigomos were performed with tail veininjected antigomo for mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p or mi R-542-3p, or mixture of antigomos for the above 4 mi RNAs 48 h before CLP or 24 h after CLP. Survival rate was recorded for 7 days.(2) In-vitro study: RAW264.7 cells were randomly assigned to the following 8 groups: Vehicle, LPS, LPS+LV-NC inhibitor/antigomo-NC, LPS+LV-mi R-133a-3p inhibitor/antigomo-mi R-133a-3p, LPS+LV-mi R-141-3p inhibitor/antigomo-mi R-141-3p, LPS+LV-mi R-3074-2-3p inhibitor/antigomo-mi R-3074-2-3p, LPS+LV-mi R-542-3p inhibitor/antigomo-mi R-542-3p and LPS+LV-mi R-mix inhibitor/antigomo-mi Rmix groups. An in-vitro sepsis model was induced by 100 ng/ml LPS stimulation for at least 2 h. Lentivirus carrying LV3-mmu-mi R-133a-3p-inhibitor, LV3-mmu-mi R-3p-inhibitor, LV3-mmu-mi R-3074-2-3p-inhibitor or LV3-mmu-mi R-542-3p-inhibitor expression plasmid were infected to RAW264.7 cells 72 h before LPS stimulation,and antigomos for 4 mi RNAs were transfected to the cells 12 h before LPS stimulation. Cell culture supernatants were harvested for determination of inflammatory cytokines TNF-? and IL-1?.Results1) We first observed significant increases of IL-1? and TNF-? in the culture supernatant from cells after LPS stimulation(P < 0.05). The 0.5 MAC isoflurane in 60% oxygen significantly decreased levels of IL-1? and TNF-? in cell culture supernatant when compared to those from LPS-stimulated cells(P < 0.05). Lentiviral over-expression of mi R-133a-3p or mi R-3074-2-3p significantly increased TNF-? level in the culture supernatant from LPS-stimulated cells with treatment of 0.5 MAC isoflurane in 60% oxygen(P < 0.05), and lentiviral over-expression of mi R-542-3p significantly increased IL-1? level in the culture supernatant from LPS-stimulated cells with treatment of 0.5 MAC isoflurane in 60% oxygen(P < 0.05). Mimic for mi R-133a-3p or mi R-3074-2-3p, or mixture of mimics for all the above 4 mi RNAs significantly increased TNF-? level in the culture supernatant from LPS-stimulated cells with treatment of 0.5 MAC isoflurane in 60% oxygen(P < 0.05). Mimic for mi R-542-3p, or mixture of mimics for all the above 4 mi RNAs significantly increased IL-1? level in the culture supernatant from LPS-stimulated cells with treatment of 0.5 MAC isoflurane in 60% oxygen(P < 0.05). While antigomo for mi R-133a-3p or mi R-3074-2-3p, or mixture of antigomos for all the above 4 mi RNAs further decreased TNF-? level in the culture supernatant from LPS-stimulated cells with treatment of 0.5 MAC isoflurane in 60% oxygen(P < 0.05), antigomo for mi R-542-3p, or mixture of antigomos for all the above 4 mi RNAs, further decreased IL-1? level in the culture supernatant from LPS-stimulated cells with treatment of 0.5 MAC isoflurane in 60% oxygen(P < 0.05). In addition, LPS stimulation induced an increase of phosphorylated NF-?B p65 protein in total cell, NF-?B p65 subunit protein in nucleus and a decrease of NF-?B p65 subunit protein in cytoplasm, indicating that LPS induced activation of NF-?B signalling pathway. The 0.5 MAC isoflurane in 60% oxygen inhibited activition of NF-?B signalling after LPS stimulation. Lentiviral over-expression of mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p or mi R-542-3p, or lentiviral over-expression of all these 4 mi RNAs partly reversed inhibition on LPS-induced NF-?B signalling activition by 0.5 MAC isoflurane in 60% oxygen, with lentiviral over-expression of all the 4 mi RNAs being the most effective intervention.In animal study, the survival rate of mice was decreased after CLP(P < 0.05 vs. sham+air group). The 0.5 MAC isoflurane in 60% oxygen significantly increased the 7-day survival rate(P < 0.05 vs. CLP+air group), which was reversed by lentiviral over-expression of all 4 mi RNAs, or lentiviral over-expression of mi R-141-3p alone(P < 0.05).2) Based on the above results, we proposed that inhibition to mi RNAs would be benefial to sepsis. Here we observed that pretreatment with lentivirus-based inhibitor for mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p or mi R-542-3p alone, increased the 7-day survival rate of CLP-challenged animals, but without a statistical significance when compared with those from CLP group, while pretreatment with mixture of lentivirus-based inhibitors for all these 4 mi RNAs significantly increased the 7-day survival rate of CLP-challenged animals(P < 0.05). We also observed that pretreatment with mixture of antigomos for all the 4 mi RNAs statistically significantly improved the 7-day survival rate of CLP-stimulated animals(P < 0.05), and treatment with antigomo for mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p or mi R-542-3p alone or mixture of antigomos for all the 4 mi RNAs improved the 7-day survival rate of CLP-challenged animals but without a statistical significance.We further observed that in LPS-stimulated RAW264.7 cells, lentivirus infection of inhibitors for mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p, or mi R-542-3p alone, or especially a combination of all these 4 mi RNAs, reduced levels of IL-1? and TNF-? in culture supernatant(P < 0.05). Similar results were obtained in LPS-stimulated cells with pretreatment of antigomos for mi RNAs.ConclusionAccording to the above-mentioned, mi RNAs mediated NF-?B inflammatory pathways contribute to the protective effects on sepsis by combination administration of 0.5MAC isoflurane with 60% oxygen; and mi R-133a-3p, mi R-141-3p, mi R-3074-2-3p and mi R-542-3p may be newer tragets for developing measures for treatment of sepsis. |
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