Citrulline Supplementation Has Protective Effects On Liver Injury In The Early Stages Of Sepsis

Part 1 Effects of citrulline supplementation on liver injury and Cit-NO cycle in septic ratsBackground and Objective:The concentration of arginine in plasma and muscle decreased in sepsis patients. However, clinical randomized controlled trials showed arginine supplementation had no protected effects on sepsis. Citrulline(Cit) involved in arginine metabolism by the way of Cit-NO cycle. And the liver is one of the organs susceptible to organ damage in sepsis as well as involved in Cit-NO cycle. The aim of this study was to investigate the effects of Cit supplementation on liver injury and Cit-NO cycle in septic rats.Methods: The cecal ligation and puncture(CLP) model was used in our study. Rats were divided into Normal group, Sham group, Cit group, Normal saline group, CLP group and CLP+Cit group. The CLP group and CLP+Cit group were then divided into 6h, 12 h, 24 h and 48 h subgroups respectively, according to the time points of sacrifice after surgery. Intragastric administration of Lcitrulline was applied to rats in Cit and CLP+Cit groups before surgery, at a dose of 200mg/d.The general condition and survival status of rats were observed every 6 hours after surgery. Rats of each group were sacrificed at predetermined time points. Rat blood and liver tissues were collected, and HE staining was used to observe the pathological changes in liver tissues. The AST and ALT levels in serum and the levels of SOD, MDA, NO and i NOS in the liver tissues were measured in each group. The concentrations of Cit and Arg in plasma were assessed by HPLC-MS/MS method. We analyzed the related data by SPSS16.0 statistical software, setting P<0.05 as statistically different.Results: The survival rates of Sham group, Cit group and NS group were all 100%(8/8). The survival rates of CLP 6h, 12 h, 24 h and 48 h subgroups were 75%(9/12), 50%(8/16), 33.33%(8/24) and 6.25%(2/32), respectively. And the survival rates of CLP+Cit 6h, 12 h, 24 h and 48 h subgroups were 83.33%(10/12), 68.75%(11/16), 66.67%(16/24) and 25%(8/32), respectively. The survival rates of CLP+Cit 24 h and 48 h subgroups were significantly higher than that of CLP 24 h and 48 h subgroups(both P<0.05). HE staining results showed that liver tissues of CLP group and CLP+Cit group appeared pathological changes of different degrees of liver damage.In addition, the serum AST levels in CLP+Cit 12 h and 24 h subgroups was significantly lower than that in CLP 12 h and 24 h subgroups(both P<0.05). At the three time points of 6h, 12 h and 24 h, the SOD activities of liver tissues in CLP+Cit groups were significantly higher than that in CLP subgroups(all P<0.05). In contrast, the MDA levels of liver tissues in CLP+Cit groups were lower than CLP groups, wherein the MDA levels in CLP+Cit 12 h and 24 h subgroups were significantly lower than CLP 12 h and 24 h subgroups( both P<0.01).At the three time points, the NO levels in CLP groups were significantly higher than CLP+Cit groups(all P <0.05). However, there was no significant difference in i NOS activities between CLP groups and CLP+Cit groups(all P>0.05). In addition, during the postoperative 24 hours, the Arg plasma level in CLP group decreased with time. Plasma concentration of Arg in CLP 24 h subgroup was significantly lower than that in the normal group(P <0.01). In our study, citrulline supplementation remarkably increased plasma concentrations of Arg and Cit in CLP+Cit groups compared with CLP groups. In addition, plasma Arg level in CLP+Cit group did not changed significantly after surgery. The concentration of Arg in CLP+Cit 24 h subgroup was close to the baseline(Cit group).Conclusion: Citrulline supplementation improved the survival rates in septic rats. Citrulline supplementation raised the activities of SOD in the liver, which improved the antioxidant capacity in septic rats. Citrulline supplementation decreased the MDA levels and reduced the extent of liver damage in the early stages of sepsis. In addition, citrulline supplementation decreased the production of NO, increased plasma Arg concentrations and remained it at the baseline in septic rats.Part 2 Effects of citrulline supplementation on cytokines and chemokines expressions in the early stages of sepsisBackground and Objective: Cytokines played an important role in the development of sepsis. In the early stages of sepsis, the body was at a proinflammatory stage with a release of a large number of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, etc. These pro-inflammatory cytokines could activate the cytokine cascade reaction and lead to the “cytokine storm”, aggravating the inflammatory and immune response and resulting in liver injury eventually. Our previous study found that citrulline supplementation could reduce the expression of IL-6, and was considered a potential immunomodulator in sepsis. Based on the previous research, the aim of this study was to further explore the effects of citrulline supplementation on the expressions of cytokines and chemokines in the early stages of sepsis.Methods: The CLP model was used in our study. Rats were divided into four groups: Normal group, Cit group, CLP group and CLP+Cit group. The CLP group and CLP+Cit group were then divided into 6h, 12 h and 24 h subgroups respectively, according to the time points of sacrifice after surgery.Luminex 1iquid chip was used to measure the serum concentrations of 27 kinds of cytokines and chemokines including EGF, Eotaxin, Fractalkine, G-CSF, GMCSF, GRO/KC/CINC-1, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-17 A, IL-18, IP-10, Leptin, LIX, MCP-1, MIP-1α, MIP-2, RANTES, TNF-α and VEGF.Results: The standard curves and quality control parameters of liquid chip results were satisfactory. Serum concentrations of all 27 kinds of cytokines and chemokines were higher in CLP 6h group than the normal group, of which 21 kinds of cytokine and chemokine increased significantly(P<0.05). Serum concentrations of the remaining 6 kinds of cytokines and chemokines IL-13, Fractalkine, LIX, IL-18, VEGF and IL-12(p70) were also higher in CLP 6h group, but without statistical significances.In the first 24 hours after surgery, the concentrations of 20 kinds of cytokines and chemokines peaked earlier in CLP group than in CLP+Cit group, including EGF, Eotaxin, G-CSF, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IFN-γ, IL-17, Leptin, MCP-1, MIP-1α, MIP-2, TNF-α, VEGF, IL-13 and Fractalkine. Only 2 cytokines GM-CSF and IP-10 peaked earlier in CLP+Cit group.In addition, concentrations of TNF-α, IL-6 and IL-1β were significantly lower in the CLP+Cit 6h subgroup than in the CLP 6h subgroup. Moreover, in 24 h after surgery, serum concentrations of IL-4 and IL-10 in the CLP+Cit 24 h subgroup were about 5 times higher than that in the CLP 24 h subgroup. Serum IL-4 concentration of CLP+Cit 24 h subgroup was significantly higher than that of CLP 24 h subgroup(P<0.05).Conclusion: Luminex results indicated that our modeling was satisfactory. Citrulline supplementation postponed the release of cytokines and chemokines in the early stages of sepsis. Citrulline supplementation inhibited the “proinflammatory state” and reduced the release of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in the early stages of sepsis. Citrulline supplementation postponed the release of IL-4 and IL-10 in sepsis.Part 3 Effects of citrulline supplementation on STAT1/STAT3 signaling pathway in septic ratsBackground and Objective: JAK/STAT signaling pathway was the common signaling pathway of a variety of inflammatory cytokines and played a pivotal role in the process of sepsis. Many studies have reported that STAT1 and STAT3 were involved the signal transduction of many inflammatory cytokines in sepsis. Based on the previous research, the aim of this study was to explore the effects of citrulline supplementation on STAT1/STAT3 signaling pathway in septic rats.Methods: The CLP model was used in our study. Rats were divided into CLP group and CLP+Cit group. Both groups were then divided into 6h, 12 h and 24 h subgroups respectively, according to the time points of sacrifice after surgery.The expression levels of STAT1 and STAT3 m RNA in rat liver tissues of each group were measured using real-time PCR method. The protein expressions of STAT1 and STAT3 were detected through western blot method. The phosphorylation levels of STAT1 and STAT3 were also detected through western blot method.Results: At the three time points of 6h, 12 h and 24 h, there were no significant differences in STAT1 and STAT3 m RNA expressions of liver tissues between CLP group and CLP+Cit group(P>0.05). Likewise, there were no significant differences in the protein expressions of STAT1 and STAT3 between CLP group and CLP+Cit group(P> 0.05). However, the phosphorylation levels of STAT1 and STAT3 in CLP+Cit 6h and 12 h subgroups were significantly lower than that in CLP 6h and 12 h subgroups(P <0.05).Conclusion: In CLP-induced septic rats, citrulline supplementation had no effect on m RNA and protein expressions of STAT1 and STAT3. However, citrulline supplementation reduced the phosphorylation levels of STAT1 and STAT3 in the early stages of sepsis.