Ameliorating Effect And Mechanism Of Vitamin K2 On Ulcerative Colitis Mice

Objective:Ulcerative colitis(UC)is a non-specific chronic inflammation of the gut,the pathogenesis of which is still unclear.Currently,there is no specific treatment for UC.Vitamin K(VK)deficiency is widespread in people with inflammatory bowel disease.Vitamin K(including Vitamin K1 and Vitamin K2)is a fat-soluble vitamin that helps to maintain the body’s metabolism and health.Vitamin K2(VK2)has higher biological activity than vitamin K1(VK1).The roles of VK2 in inhibiting inflammation,oxidative stress and suppressing immune response have been reported.The aim of this study is to establish a mouse model of ulcerative colitis induced by dextran sulfate sodium(DSS)and investigate the amelioration effect and mechanism of various doses of VK2(Menaquinone-4,MK-4)on UC in mice.Methods:Different concentrations of DSS solution were given to establish the mouse model of UC.Forty C57BL/6J mice were randomly divided into four groups after one week of acclimatization feeding.The groups were as follows: control group(CON),2.0% DSS group(DL),2.5% DSS group(DM)and 3.0% DSS group(DH).All the mice were fed with an ordinary diet.The CON group drank tap water freely,while the DL,DM and DH groups were given 2.0%,2.5% and 3.0% DSS solution,respectively.The whole experiment lasted for seven days.The disease activity index(DAI)was calculated from daily data of body weight,fecal condition,and blood in stool.The mice were humanly euthanized at the end of the study.The blood,spleen,and colon tissues were collected,and stored at-80°C for future analyses.The colonic histopathology,oxidative stress indexes and inflammatory cytokines were analyzed.The amelioration effect of VK2 supplementation on UC in mice was then investigated.Sixty C57BL/6J mice were randomly divided into four groups after one week of acclimatization feeding.The groups were as follows: control group(CON),model group(DSS),15 mg/kg·bw VK2 group(LVK2),30 mg/kg·bw VK2 group(MVK2),60 mg/kg·bw VK2 group(HVK2)and 5-aminosalicylic acid group(5-ASA).All mice were fed with an ordinary diet.The CON group drank tap water freely and received intragastric administration of corn oil for 14 days.The DSS group drank 3.0%DSS solution for 7 days,followed by the intragastric administration of corn oil for 14 days,and 1.5% DSS solution from Day 15 to 21.The LVK2,MVK2,and HVK2 groups received intragastric administration of 15,30,or 60 mg/kg·bw of VK2(dissolved in corn oil)for 14 days.The 5-ASA group received intragastric administration of 100 mg/(kg·bw)5-ASA for 14 days.The other treatment was the same as the DSS group.The colonic histopathology,serum MK-4/7,oxidative stress indexes and inflammatory cytokines were analyzed.The expression of nuelear factor-kappa B(NF-κB)pathway-related proteins were measured using western blot(WB).Serum concentrations of diamine oxidase(DAO)and colonic epithelial tight junction proteins were analyzed to investigate the protective effect of VK2 on the intestinal mucosal barrier in UC mice.The gut microbiota and short-chain fatty acids(SCFAs)were measured by 16 S r RNA and targeted metabolomics to explore the regulatory effect of VK2 on intestinal flora in UC mice.Results:The mouse model of DSS-induced UC was successfully established.The mice in the DH group showed more significant ulcerative symptoms,higher histological scores and greater changes in inflammatory cytokines and oxidative stress levels than the other groups.Compared with the DSS group,the DAI of mice in the LVK2,MVK2 and HVK2 groups were decreased by 21.74%,23.91%,and 45.65%,respectively(all P < 0.05),and the colon lengths were increased by 1.94%,17.78%,and 11.67%,respectively(all P <0.05).The serum MK-4 levels in the DSS and CON groups were similar,while it was significantly lower than those in the LVK2,MVK2 and HVK2 groups(all P < 0.05).Meanwhile,the serum MK-7 levels were lower in the DSS group than those in the CON,MVK2,and HVK2 groups(all P < 0.05).Compared with the DSS group,the interleukin1β(IL-1β)levels in the LVK2,MVK2 and HVK2 groups were decreased by 66.90%,74.74%,and 45.42%,respectively(all P < 0.05),and the interleukin 10(IL-10)levels were increased by 89.03%,84.96%,and 93.06%(all P < 0.05).Additionally,the tumor necrosis factor-α(TNF-α)levels in the MVK2 and HVK2 groups was reduced by 64.44%and 36.25%(P < 0.05)in comparison to the DSS group.Western blot analysis revealed that VK2 intervention significantly ameliorated the changes in NF-κB pathway-related proteins caused by DSS induction(P < 0.05).The intestinal barrier proteins results showed that the expression levels of colonic mucins 2(Muc2),Occludin,and zonula occluden 1(ZO-1)in the MVK2 group were significantly higher than that in the DSS group(P < 0.05),and the expression levels of colonic Muc2 and Occludin in the HVK2 group were significantly higher than that in the DSS group(P < 0.05).In addition,the supplementation of medium-and high-dose of VK2 showed a positive effect on oxidative stress.Compared with the DSS group,the medium-dose VK2 intervention had a significant ameliorative effect on intestinal flora alteration,and increased the relative abundance of SCFA-producing bacteria,such as Eubacterium＿ruminantium＿group and Faecalibaculum.Correlation analysis showed that changes in gut microbiota were significantly correlated with inflammatory cytokines and intestinal barrier proteins.Also,the results of Veillonella,an MK-7 producing bacterium,showed a difference between the HVK2 and DSS groups.Conclusion:The current study successfully established a mouse model of DSS-induced ulcerative colitis,which showed damage to the intestinal mucosa and abnormality in the gut microbiota.The doses of 15 mg/(kg·bw),30 mg/(kg·bw)and 60 mg/(kg·bw)VK2 all ameliorated the damage of ulcerative colitis in mice,and 30 mg/(kg·bw)VK2 showed the best efficacy in reducing inflammation and oxidative stress,improving intestinal barrier function,and improving gut microbiota.The different effects of various doses of VK2 may be related to their differential regulation of gut microbiota composition.These findings may provide a theoretical basis for the use of VK2 as an adjuvant therapy for UC.Future clinical studies are warranted to confirm these findings in humans.