Study On The Effect Of Lactobacillus Acidophilus On Calcium Sensitization Pathway Of Intestine In Mice After Severe Head Injury

The intestinal motility disorder would arise after severe head injury(SHI)in early stage,and then would affect the outcome and prognosis of the injuried seriously.The early manifestation of intestinal motility deficiencywill lead to gastroesophageal reflux,vomiting,abdominal distension,constipation and other feeding intolerance symptoms,and it would lead to malnutrition,aspiration,sepsis and even multiple organdysfunction syndrome(MODS)and other serious complications in late.Therefore the remedy of the intestinal motility deficiencyafter severe head injury is conducive to the recovery and beneficial to prognosis of the disease.The basic unit of the small bowel movement is smooth muscle cells(SMC).20 k D of myosin light chain(MLC20)is an important structural molecule among them that regulates the contraction of smooth muscles.After phosphorylation at serine(Ser)19 of MLC20,the relative displacement between the thick and thin filaments isinduced,and the cells contraction would occur.Basic researches suggested that the phosphorylation of MLC20 is regulated by multiple signaling pathways,however,the calcium dependent / calcium sensitization pathwaysweretwomain pathways.Phosphorylation of myosin phosphatase target subunit 1,the functional structure of myosin light chain phosphatase(MLCP),plays the key role of calcium sensitization pathway,which is regulated by Rho-associated kinase(ROCK)and Zipper-interacting protein kinase(ZIPK)in the upper reaches of the pathway,and participates in the occurrence and development of a variety of gastrointestinal motility disorders.However,whether the pathway is associated with intestinal motility deficiency after severe brain injury,is still not clear.So far,there still lack spreventive clinical treatments of severe patients with severe intestinal dynamic problems.Lactobacillus acidphilus(La)can be auxiliary used in the prevention and improvement of gastrointestinal motilitydeficiency caused by functional gastrointestinal diseases,operation,infection and so on.Previous studies found out that besides improvingthe intestinal motility deficiencyafter severe head injury,Lactobacillus acidophilus can also adjust the damageof the intestinal muscle contraction frequency,amplitude and tension.Therefore,it'sagreat significance to explore the mechanism of the above effects in clinical application.However,the molecular mechanisms by which probiotic Lactobacillus modulates intestinalmotility in SHI mouse model have not been explored.Combined with previous studies and related literature,we hypothesize that: severe head injury might lead to the damage of the structure of intestinal smooth muscle cells,result in abnormalconduction of calcium sensitivationpathway,bring about lack of intestinal motility further more.The mechanism how Lactobacillus acidophilusmakes effects may be related to repairedamaged smooth muscle cells,and regulate related signal transduction of calcium sensitivationpathway in addition.In the present study,we aim to investigate the molecular mechanism of intestinal motility deficiency induced by severe brain injury,and choose one of the most important probiotics,Lactobacillus acidophilus,to observe the role of the key molecules in the regulation of gastrointestinal motility,so that to clarify the effect of severe trauma on gastrointestinal function and how probiotics effects,furthermore,provide the basis for the application of probiotics tothe clinicalpractice in severe trauma area.?Materials and methods1.Establishment of SHI model 90 healthy male adult C57bl/6 miceweredivided into 3 groups,including sham injury group(control group),injury group(SHI+MRS group),La group(SHI+La group),randomly.There were three time points 1d,3d and 7d respectively in each group.The mice model of severe head injury was built up by an improved Feeney's weight-dropping method,which modified by Huan Fang et al.Meanwhile,the sham injury group only opened the bone window without hit.Group La would dissolve in the medium by gavage 6h after injury,each mouse feeding amount is not lower than 1x1010cfu/d,as the same time,group sham and group injury were feed the same capacity of MRS.All mice had the freedom to get the diet and water for the rest of the time.2.The pathological changes of small intestine morphology and structure were observed afterHE staining of the terminal ileum tissue.Meanwhile the thickness of the smooth muscle was measured.3.The expression and distribution of MLC20,p-MLC20,MLCP and p-MYPT1 subunits were detected by immunohistochemical staining with specific biotin labeling.4.The expression of MLCP in mouse ileum was detected by enzyme linked immunosorbent assay.5.The expression of ROCK and ZIPK gene were detected by RT-PCR method.?Results1.Alternation of pathological in small intestine and muscle layer thicknessHE staining showed that,the small intestine were structural integrity in sham injury group.Tissues of SHI group mice in 1d,3d,7d obviously showed pathological changes.In the 7th day,intestinal mucosa epithelial showed focal necrosis,ulceration,and seriously damage on the intestinal mucosal barrier.Meanwhile,the muscle layer thickness became thiner(vs.sham injury group,1d,3d P<0.05,7d P<0.01).In the 7th day of Lactobacillus acidophilus intervention,the morphology,height,and middle diameter of small intestinal villus were no significant difference compared with sham injury group,and no edema and inflammatory reaction in muscle layer.There were no obviously pathological and thickness alternation.2.Alternation of MLC20 and its phosphorylation levelImmunohistochemical results showed that the expression of MLC20 was no significant difference between the three groups.p-MLC20 level in SHI after 1d,3d,7d were significantly decreased(vs.sham injury group,P<0.05).The intervention of Lactobacillus acidophilus after 1d,3d,7d,the level of p-MLC20 were significantly increased(vs.SHI group,P<0.05).3.Alternation of MLCP expression and tissue distributionELISA results showed that MLCP expression in SHI group after 1d,3d,7d levels were significantly increased(vs.sham injury group,P<0.01).The intervention of Lactobacillus acidophilus after 1d,3d,7d,the expression of MLCP decreased significantly(vs.SHI group,P<0.05).Immunohistochemistry results were consistent with ELISA results.4.Alternation of p-MYPT1 distributionImmunohistochemistry results showed that p-MYPT1 distribution in SHI after 1d,3d,7d were decreased(vs.sham injury group,P<0.01).The use of Lactobacillus acidophilus in the day of 1d,3d,7d,the distribution of p-MYPT1 was obviously increased(vs.SHI group,P<0.01).5.Alternation of ROCK mRNAPCR results showed that 1d,3d,7d,ROCK m RNA were significantly reduced(vs.sham injury group,P<0.01)in SHI group.Use of Lactobacillus acidophilus after 1day,ROCK mRNA were increased significantly(vs.SHI Group,P<0.05);after 3days and 7 days intervention by Lactobacillus acidophilus,ROCK mRNA were increased(vs.SHI group,P<0.01).6.Alternation of ZIPK mRNAPCR results showed that 1d,3d,7d,ZIPK m RNA were significantly reduced(vs.sham injury group,P<0.01)in SHI group.Use of Lactobacillus acidophilus after 1day and 3days,ZIPK mRNA were increased significantly(vs.SHI Group,P<0.05);after 7days intervention by Lactobacillus acidophilus,ZIPK mRNA were increased(vs.SHI group,P<0.01).In the La group,the difference was statistically significant(P<0.05)at different time points.?conclusions1.The phenomenon of the arrangment disorder,degeneration,nuclear condensation of smooth muscle cells,inflammatory cell infiltration,interstitial edema,would turn upin small intestinal tissue of miceafter severe head injury.At the same time,the small intestinal smooth muscle layer becomes thinner and the number of cellsdecreased.2.The p-MYPT1 levels decreased,MLCP expression and activity increased,intestinal calcium sensitivationpathway was abnormallyactivated,MLC20 phosphorylation levels decreased,while the ROCK and ZIPK gene expression levels decreased in small intestinal tissue of mice that after severe head injury.3.The pathological state of the small intestine was improved,the degree of smooth muscle cells damage was repaired,and the thickness of the muscular layer was increased after feeding the mice after severe head injury with Lactobacillus acidophilus.4.Lactobacillus acidophiluscould increase the ROCK and ZIPK gene level,raise the phosphorylation of MYPT1,reduce the expression and distribution of MLCP,inhibit the excessive activation of intestinal calcium sensitivationpathway,and maintain the level of MLC20 phosphorylationin mice after severe brain injury.5.Lactobacillus acidophiluscould improve the intestinal motility deficiency after severe head injury,which might be related to the regulation of calcium sensitivation pathway that mediated by ROCK and ZIPK.