嗜酸乳杆菌改善创伤性脑损伤小鼠肠道平滑肌收缩功能的作用及可能机制

Role and molecular mechanism of Lactobacillus acidophilus in improving intestinal smooth muscle contraction in mouse with traumatic brain injury

目的初步探讨嗜酸乳杆菌改善创伤性脑损伤(TBI)小鼠肠道平滑肌收缩功能的作用及可能机制。方法按照随机数字表法将90只C57BL/6雄性小鼠分为假伤组、TBI组、TBI+嗜酸乳杆菌组,每组30次。TBI组、TBI+嗜酸乳杆菌组采用改良的Feeney自由落体撞击法建立TBI后肠动力不足模型,假伤组只开颅骨骨窗不进行打击。假伤组及TBI组灌胃0.5 ml嗜酸乳杆菌培养基,TBI+嗜酸乳杆菌组灌胃0.5 ml嗜酸乳杆菌混悬液(约含菌1×1010CFU)。各组每天灌胃1次,其余时间自由饮食水。分别在伤后1,3,7 d取末端回肠组织(距离盲肠1.5 cm),免疫组化染色检测磷酸化20 kDa肌球蛋白轻链(p-MLC20)水平,ELISA法检测肌球蛋白轻链激酶(MLCK)活性及L型电压依赖性钙离子通道α1C亚基(Cav1.2)、三磷酸肌醇受体(IP3R)、兰尼碱受体3(RyR3)蛋白表达。结果 (1)TBI组1,3,7 d的p-MLC20水平为(530.6±101.5)ng/ml、(566.8±86.9)ng/ml、(635.2±129.6)ng/ml,较假伤组的(813.7±148.9)ng/ml、(802.6±151.2)ng/ml、(805.5±139.9)ng/ml显著降低(P均<0.05);而TBI+嗜酸乳杆菌组1,3,7 d的p-MLC20水平为(790.7±59.4)ng/ml、(769.8±85.4)ng/ml、(731.8±82.9)ng/ml,显著高于TBI组(P均<0.05)。(2)TBI组1, 3, 7 d的MLCK活性为(29.4±5.0)U/L、(31.2±3.4)U/L、(30.7±2.4)U/L,明显弱于假伤组的(44.9±6.1)U/L、(44.6±1.7)U/L、(45.1±3.7)U/L(P均<0.05);TBI+嗜酸乳杆菌组1,3,7 d的MLCK活性为(35.2±3.1)U/L、(38.7±3.9)U/L、(34.7±2.9)U/L,较TBI组明显增强(P均<0.05)。(3)TBI组1, 3, 7 d的Cav1.2表达量为(1.7±0.4)ng/L、(2.3±0.4)ng/L、(2.9±0.5)ng/L,明显低于假伤组的(5.8±0.6)ng/L、(5.6±0.6)ng/L、(5.7±0.7)ng/L (P均<0.05);TBI+嗜酸乳杆菌组1,3,7 d的Cav1.2水平为(2.8±0.6)ng/L、(4.7±0.6)ng/L、(4.9±0.5)ng/L,较TBI组明显升高(P均<0.05)。TBI组1, 3, 7 d的IP3R表达量为(12.4±2.5)μg/L、(15.7±3.0)μg/L、(16.3±3.1)μg/L,明显低于假伤组的(30.3±3.0)μg/L、(31.9±2.6)μg/L、(32.1±1.7)μg/L(P均<0.05);TBI+嗜酸乳杆菌组1 d的IP3R表达量为(13.1±1.9)μg/L,与TBI组的(12.4±2.5)μg/L比较,差异无统计学意义(P>0.05);TBI+嗜酸乳杆菌组3 d和7 d的IP3R表达量为(18.4±2.4)μg/L、(22.9±2.8)μg/L,较TBI组明显升高(P均<0.05)。TBI组1,3,7 d的RyR3表达量为(30.8±4.4)pg/ml、(29.1±3.6)pg/ml、(27.9±2.9)pg/ml,明显低于假伤组的(43.5±3.2)pg/ml、(44.9±2.9)pg/ml、(44.2±2.0)pg/ml (P均<0.05);TBI+嗜酸乳杆菌组1,3,7 d的RyR3表达量为(33.3±2.5)pg/ml、(30.4±2.3)pg/ml、(30.2±2.4)pg/ml,与TBI组比较,差异无统计学意义(P均>0.05)。结论嗜酸乳杆菌可提高TBI小鼠肠道平滑肌p-MLC20水平,增强MLCK活性,促进Cav1.2、IP3R、RyR3表达,从而保证钙依赖性通路信号的正常传导,可能是其改善TBI小鼠肠道平滑肌收缩的机制之一。

Objective To primarily explore the ffct of Lactobacillus acidophilus in improving the intestinal smooth muscle contraction in mice with traumatic brain injury(TBI)and its possible mechanism.Methods A total of 90 C57BL/6 male mice were divided into sham group,TBI group and TBI+Lactobacillus acidophilus group according to the random number table,with 30 rats per group.The TBI group and the TBI+Lactobacillus acidophilus group were conducted TBI procedure by modified Feeney method.The sham group underwent craniotomy without brain injury.The sham group and the TBI group were gavaged with 0.5 ml MRS culture medium,the TBI+Lactobacillus acidophilus group was gavaged with 0.5 ml Lactobacillus acidophilus(about 1×10^10CFU).Each group was gavaged once daily with access to food and water ad libitum.The terminal ileum segments(1.5 cm from caecum)were taken at days 1,3 and 7 after TBI.The levels of the phosphorylation of myosin light chain(p-MLC20)were detected by immunohistochemical staining.The activity of myosin light-chain kinase(MLCK)and the levels of the L-type voltage-gated calcium channel alc-subunit(Cavl.2),inositol 1,4,5-trisphosphate receptor(IP3R)and ryanodine receptor3(RyR3)were measured by ELISA method.Results(1)The levels of p-MLC20 in TBI group were respective(530.6±101.5)ng/ml,(566.8±86.9)ng/ml,(635.2±129.6)ng/ml at days 1,3 and 7,showing significant decreases in comparison with those in sham group[(813.7±148.9)ng/ml,(802.6±151.2)ng/ml,(805.5±139.9)ng/ml](all P<0.05).The levels of p-MLC20 in TBI+Lactobacillus acidophilus group were respective(790.7±59.4)ng/ml,(769.8±85.4)ng/ml,(731.8±82.9)ng/ml at days 1,3 and 7,showing significant raises in comparison with those in TBI group(all P<0.05).(2)The activity of MLCK in TBI group were respective(29.4±5.0)U/L,(31.2±3.4)U/L,(30.7±2.4)U/L at days 1,3 and 7,showing significant differences in comparison with that in sham group[(44.9±6.1)U/L,(44.6±1.7)U/L,(45.1±3.7)U/L](allP<0.05).The activity of MLCK in TBI+Lactobacillus acidophilus group were respective(35.2±3.1)U/L,(38.7±3.9)U/L,(34.7±2.9)U/L at days 1,3 and 7,showing significant differences in comparison with that in TBI group(all P<0.05).(3)The levels of Cav1.2 in TBI group were respective(1.7±0.4)ng/L,(2.3±0.4)ng/L,(2.9±0.5)ng/Lat days 1,3 and 7,showing signifcant decreases in comparison with those in sham group[(5.8±0.6)ng/L,(5.6±0.6)ng/L,(5.7±0.7)ng/L](all P<0.05).The levels of Cav1.2 in TBI+Lactobacillus acidophilus group were respective(2.8±0.6)ng/L,(4.7±0.6)ng/L,(4.9±0.5)ng/L at days 1,3 and 7,showing significant raises in comparison with those in TBI group(all P<0.05).The levels of IP3R in TBI group were respective(12.4±2.5)μg/L,(15.7±3.0)μg/L,(16.3±3.1)μg/L at days 1,3 and 7,showing significant decreases in comparison with those in sham group[(30.3±3.0)μg/L,(31.9±2.6)μg/L,(32.1±1.7)μg/L](all P<0.05).There was no signifcant difference between the levels of IP3R at days 1 in TBI+Lactobaeillus acidophilus group[(13.1±1.9)μg/L]and in TBI group[(12.4±2.5)μg/L](P>0.05).The levels of IP3R in TBI+Lactobacillus acidophilus group were(18.4±2.4)μg/L and(22.9±2.8)μg/L at days 3 and 7,showing significant raises in comparison with those in TBI group(all P<0.05).The levels of RyR3 in TBI group were respective(30.8±4.4)pg/ml,(29.1±3.6)pg/ml,(27.9±2.9)pg/ml at days 1,3 and 7,showing significant decreases in comparison with those in sham group[(43.5±3.2)pg/ml,(44.9±2.9)pg/ml,(44.2±2.0)pg/ml](all P<0.05).The levels of RyR3 in TBI+Lactobacillus acidophilus group wererespective(33.3±2.5)pg/ml,(30.4±2.3)pg/ml,(30.2±2.4)pg/ml at days 1,3 and 7,showing no significant differences from those in TBI group(all P>0.05).Conclusions Lactobacillus acidophilus can increase p-MLC20 levels,enhance MLCK activity and improve the expressions of Cav 1.2,IP3R and RyR3,which subsequently ensure the normal conduction of Ca^2+-dependent pathway.It may be one of the mechanisms Lactobacillus acidophilus improving the intestinal smooth muscle contraction in mice with TBI.