Studies Of The Effects Of Lactobacillus Acidophilus On The Ca²⁺-Dependent Pathways Of The Small Intestinal Smooth Muscle In SHI Mouse Model

The case fatality rate of severe head injury（SHI）is as high as 20-50%.Intestinal motility deficiency（IMD）is a common complication after SHI.If not effectively prevented or treated in early time,it may cause intestinal flora disturbance,exacerbate the inflammatory response,and even cause multiple organ dysfunction syndrome（MODS）.Now the drugs for IMD used in clinical practice have some side effects.With the startup of “the human intestinal macro genome project”,the role of probiotics in human disease therapy has been concerned widely in recent years.Studies have shown that probiotics could not only regulate intestinal flora imbalance,but also improve the intestinal motility.However,most of the studies have just observed the effect of using probiotics without exploring its mechanisms.The contraction of smooth muscle cells is the end effector of intestinal motility,and Ca²⁺-dependent pathway plays an important role in regulating the smooth muscle cells’ contraction.External stimulati can increase the intracellular concentration of Ca²⁺ by the influx of extracellular Ca²⁺ through Cav1.2 and the Ca²⁺ released from the sarcoplasmic reticulum through IP3 R and RyR3.The cytosolic Ca2 + combines with the calmodulin and forming the Ca²⁺/Ca M complex.This complex activates the MLCK to phosphorylate the MLC20,leading to smooth muscle contraction.Thus,the phosphorylation levels of MLC20 have close relationship with the contraction of smooth muscle cells.Studies have found that PKC played an important role in regulating the MLC20 phosphorylation and the contraction of intestinal smooth muscle.Our research group has found that the lactobacillus acidophilus could improve the contraction of the intestinal smooth muscle of mice after SHI.On this basis this research adopted a mice model with IMD after SHI,and observed the signaling molecule changes in Ca²⁺-dependent pathway of intestinal smooth muscle in SHI mice that were gavaged with lactobacillus acidophilus for 1,3,7 days separately.We try to verify the effects of lactobacillus acidophilus on the Ca²⁺-dependent pathway,and reveal the possible mechanism of the improvement of lactobacillus acidophilus on the intestinal motility of SHI mice tentatively.This research can not only provide experimental evidence for the use of lactobacillus acidophilus in trauma areas,but also provide a reference for investigating the mechanism of probiotics improving the intestinal motility.ObjectiveTo observe the signaling molecule expression changes in Ca²⁺-dependent pathway of intestinal smooth muscle in SHI mice,and verify the effects of lactobacillus acidophilus on the Ca²⁺-dependent pathway,and reveal the possible mechanism of the improvement of lactobacillus acidophilus on the intestinal motility of SHI mice tentatively.MethodsNinety C57BL/6 mice（18-24g）were randomly divided into three groups including sham,SHI,and SHI+Lactobacillus acidophilus（SHI +La）groups（30 mice per group）.Mice in each group were divided into three time points（1 day,3 days and 7 days,10 mice/ point）.All micewereanesthetized with 10% chloral hydrate by intraperitoneal injection before experiment.The SHI procedure of mice in SHI group and SHI+La group were conducted according to the modified Feeney’s method.Mice in the sham group（n=30）underwent only opening the scalp and a craniotomy following suturing but without brain injury.The gavage was started about 4 hours after SHI and when the mouse was awake.Mice in sham group and SHI group were fed with MRS culture medium（0.5 ml each mouse per day）by gastric infusion,and mice in SHI +La group were gavaged with Lactobacillus acidophilus（1×1010CFU/d）,separately.Each mouse was gavaged one time a day and received a conventional balanced diet and water ad libitum at the rest time.The terminal ileum segments（1.5cm from caecum）of mice were taken on days 1,3,7 after SHI.The MLC20 phosphorylation（p-MLC20）was detected by Western Blotting.MLCK protein concentrations were measured by ELISA and immunohistochemistry staining,and MLCK activity was detected by ELISA.PKC、Cav1.2、IP3R、RYR3 protein concentration were measured by ELISA.Results（1）Western blotting analysis showed that the levels of MLC20 phosphorylation were significantly decreased at 1,3 and 7 days after SHI（SHI vs sham,P < 0.05 at 1,3,and 7 days）;Lactobacillus acidophilus significantly attenuated SHI-mediated inhibition of MLC20 phosphorylation（SHI+La vs SHI,P < 0.05 at 1,3 and 7 days）.（2）ELISA and immunohistochemistry analysis showed that the levels of MLCK at 1,3 and 7 days after SHI were significantly decreased（SHI vs sham,P < 0.05 at 1,3 and 7 days）.The levels of MLCK were significantly higher than that in sham after treatment with Lactobacillus acidophilus for 1,3,7 days,separately（SHI+La vs SHI,P < 0.01 at 1,3 and 7 days）.The activity of MLCK at 1,3 and 7 days after SHI was significantly weaker than that in sham group at the same time point（SHI vs sham,P < 0.01 at 1,3 and 7 days）.The activity of MLCK was significantly enhanced after treatment with Lactobacillus acidophilus for 1,3 and 7 days（SHI+La vs SHI,P < 0.05 at1,3,and 7 days）.（3）The levels of PKC at 1,3,and 7 days after SHI were significantly higher than that in sham group at 1,3 and 7 days（SHI vs sham,P < 0.05 at 1,3 and 7 days）.Treatment of SHI mice with Lactobacillus acidophilus for 1,3,and 7 days could significantly lower the expression of PKC（SHI+La vs SHI,P < 0.05 at 1,3,and 7 days）.（4）ELISA analysis showed that the levels of Cav1.2 were significantly decreased at 1,3 and 7 days after SHI（SHI vs sham,P < 0.01 at 1,3 and 7 days）.Treatment with Lactobacillus acidophilus could significantly raise the levels of Cav1.2 at 1,3 and 7 days（SHI+La vs SHI,P < 0.01 at 1,3 and 7 days）.（5）The expression of IP3 R at 1,3,and 7 days after SHI was significantly lower than that in sham group at 1,3,and 7 days（SHI vs sham,P < 0.01 at 1,3 and 7 days）.The level of IP3 R were increased after treatment with Lactobacillus acidophilus for 1 day,but were not significant when compared with the SHI group（SHI+La vs SHI,P > 0.05 at 1 day）.Treatment with Lactobacillus acidophilus for 3 and 7 days could significantly raise the expression of IP3R（SHI+La vs SHI,P < 0.01 at 3 and 7 days）.（6）ELISA analysis showed that the levels of RyR3 were significantly decreased at 1,3 and 7 days after SHI（SHI vs sham,P < 0.01 at 1,3 and 7 days）.Treatment of SHI mice with Lactobacillus acidophilus for 1,3 and 7 days could raise the levels of IP3 R,but there was no statistical significance（SHI+La vs SHI,P > 0.05 at 1,3 and 7 days）.Conclusions1.There were abnormal changes in the levels of signal molecule in Ca²⁺-dependent pathway of intestinal smooth muscle after severe head injury.2.The MLC20 phosphorylation level and the MLCK level were decreased after severe head injury,as well as the activities of MLCK.Lactobacillus acidophilus could attenuate SHI-mediated inhibition of MLCK and MLC20 phosphorylation.3.The levels of PKC were increased markedly after severe head injury.Lactobacillus acidophilus significantly attenuated SHI-mediated induction of PKC expression.4.The expression of Cav1.2,IP3 R,and RyR3 were significantly decreased after severe head injury.Lactobacillus acidophilus could attenuate SHI-mediated reduction of Cav1.2,IP3 R,and RyR3.