| Background/Purpose:Microcirculation dysfunction proved to be the main pathogenesis of neonatal necrotizing enterocolitis(NEC).Meanwhile,the current consensus indicates that hydrogen sulfide(H2S)is associated with blood vessel relaxation.What's more,activation of PPAR?/?-eNOS/NO proved to be protective in high glucose model.We hypothesize that administration of H2S can help increase intestinal microcirculation of necrotizing enterocolitis rats,and this protective effect may be associated with PPAR?/?-eNOS/NO signaling.Methods:Newborn Sprague-Dewley(SD)rats were used to induce experimental NEC by formula-feeding/hypoxia/hypothermia/Lipopolysaccharide peritoneal injection for three days.Terminal ileum of newborn rats from control group and NEC group were resected to detect the expression of CBS,CSE and 3-MST by western blotting and IHC.According to the results in the above step,we detect the expression of CBS in digestive tract of 3 days old rats by western blotting and IHC.Then,120 newborn Sprague-Dewley(SD)rats were randomly divided into 6 groups:the control group,NEC group,GYY4137 group(7.5 mg/kg.d,15 mg/kg.d,30 mg/kg.d),and AOAA group.HE stain,Histologic Injury Score and photographs were used to show the injury of intestinal tissue.Expression of CBS was detected by western blotting and Immunohistochemistry.Intestinal microvascular blood flow were monitored by laser speckle flowmetry and the survival time of the newborn rats was analyzed by SPSS.Last but not the least,100 newborn Sprague-Dewley(SD)rats were randomly divided into 5 groups:the control group,NEC group,GYY4137 group(7.5 mg/kg.d),and GSK0660 group.HE stain,Histologic Injury Score and photographs were used to show the injury of intestinal tissue.Expression of CBS,PPAR?/? and eNOS was detected by western blotting and Immunohistochemistry.Intestinal microvascular blood flow was measured by laser speckle flowmetry and the survival time of the newborn rats was analyzed by SPSS.AOAA is inhibitor of CBS,which is endogenous H2S synthetase.GYY4137 can release H2S in a slow and steady way.GSK0660 is inhibitor of PPAR?/?.Results:1.The expression of CBS in NEC group was significant decreased than in CTR group(p<0.05)but expression of CSE and 3-MST was rarely detected in both two groups by western blot and IHC.2.Expressions of CBS are both relatively high in stomach,ileum and large intestine of 3-days rats,it shows that predilection sites of NEC may has association with expression of CBS.3.Compared with control(CTR)group,intestinal microvascular blood flow of NEC rats was significant decreased yet with Histologic Injury Score and death rate of newborn rats were significant increased.NEC rats treated with7.5 mg/kg.d GYY4137 reversed all those changes.When NEC rats were treated with 15 mg/kg.d GYY4137,intestinal microvascular blood flow were further increased than in 7.5 mg/kg.d.Whereas,Histologic Injury Score and death rate in the group with 15 mg/kg.d GYY4137 had no significant difference from NEC pups.When NEC modeling pups were injected with 30 mg/kg.d GYY4137,it showed significant increase of death rate with blood stasis and necrosis of intestine.AOAA inhibited synthesis of H2S effectively and exacerbated NEC.AOAA inhibited the expression of CBS and accelerated NEC.GYY4137 inhibited the espression of CBS in a dose dependent way.Nevertheless,there is no significant difference of intestinal microvascular blood flow between the two groups.4.When NEC pups were co-injected with GYY4137 and GSK0660,it showed decreased death rate and NEC severity score compared with GYY4137 pups.However,there is no significant difference of intestinal microvascular blood flow between the two groups.Espression of CBS and eNOS were significant decreased in NEC group than control group,while PPAR?/? were significant increased.GSK0660 increased the expression of CBS and eNOS.Both GYY4137 and AOAA inhibite the expression of CBS and eNOS,but they induce the expression of PPAR?/?.Conclusion:H2S(7.5mg/kg.d)could protect intestinal microcirculation of newborn NEC pups.When its concentration increases,the side effects are also gradually rise.Inhibitor of PPAR?/? could upregulate eNOS/NO signaling to promote the protection of H2S.The mechanism was nothing to do with intestinal microcirculation. |
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