单羧酸转运蛋白1调节乳酸转运对大鼠脊髓损伤后星形胶质细胞分化的影响

Effects of monocarboxylate transporter 1 on the proliferation,migration and differentiation of astrocytes after spinal cord injury in rats

目的 探讨单羧酸转运蛋白1(MCT1)调控乳酸转运对大鼠脊髓损伤(SC1)后星形胶质细胞(AS)分化的作用及其机制.方法 成年雌性Sprague-Dawley(SD)大鼠购自山西医科大学实验动物中心,使用改良Allen's法制作SCI模型,分为假手术组、SCI(12 h)组、SCI+LA组、SCI+AZD组,每组5只,共20只,观察SCI后腹腔注射外源性乳酸钠或MCT1抑制剂对大鼠脊髓AS分化的作用.培养SD大鼠脊髓AS并建立氧糖剥夺/复氧(OGD/R)模型,首先观察不同时间点OGD/R处理AS的乳酸含量和MCT1表达.其次观察OGD/R后添加外源性乳酸钠或MCT1抑制剂对AS分化的影响,分为正常组,OGD/R(OGD4.5 h/R12 h)组,OGD/R+乳酸钠组,OGD/R+乳酸钠+AZD组.最后分析Bay 11-7082(NF-κB抑制剂)或Stattic(STAT3抑制剂)处理通路蛋白对AS分化的影响,分为OGD/R+乳酸钠组,OGD/R+乳酸钠+BAY组,OGD/R+乳酸钠+STA组.采用单因素方差分析,组间比较采用t检验.结果 SCI 12 h组大鼠脊髓MCT1表达高于假手术组(2.776±0.353比1.000,q=13.460,P<0.01).SCI+LA 组脊髓 MCT1(2.767±0.208 比 1.900±0.100,q=10.840,P<0.01)和 S100A10(1.933±0.153 比 1.630±0.061,q=5.924,P<0.05)蛋白表达高于 SCI 组,C3蛋白表达低于 SCI 组(1.603±0.035 比 1.830±0.147,q=4.561,P<0.05);而 SCI+AZD 组脊髓MCT1(1.233±0.153 比 1.900±0.100,q=8.341,P<0.05)和 S100A10(1.237±0.067 比 1.630±0.061,q=7.681,P<0.01)蛋白表达低于 SCI 组,C3 蛋白表达高于 SCI 组(2.340±0.082 比 1.830±0.147,q=10.260,P<0.01).体外培养的原代星形胶质细胞在OGD4.5 h/R12 h组MCT1蛋白表达高于正常组(1.760±0.053 比 1.000,q=6.415,P<0.01),OGD/R+LA 组 AS 胞内乳酸含量(0.028±0.001 比 0.022±0.001,q=6.415,P<0.01),MCT1(2.200±0.100 比 1.633±0.058,q=13.870,P<0.01)和 p-STAT3 蛋白(3.400±0.173 比 2.133±0.252,q=12.850,P<0.01)表达高于OGD/R 组,细胞核 NF-κB 蛋白表达低于 OGD/R 组(1.153±0.129 比 1.933±0.153,q=12.100,P<0.01),A1 分化低于 OGD/R 组(1.133±0.058 比 1.600±0.100,q=7.000,P<0.01),A2 分化高于OGD/R 组(2.300±0.173 比 1.433±0.058,q=15.920,P<0.01).而 O GD/R+LA+AZD 组 AS 胞内乳酸含量(0.014±0.002 比 0.022±0.001,q=9.278,P<0.01),MCT1(1.430±0.082 比 1.633±0.058,q=4.976,P<0.01)和 p-STAT3 蛋白(1.533±0.153 比 2.133±0.252,q=6.085,P<0.05)表达低于OGD/R组,细胞核NF-κB蛋白表达高于OGD/R组(2.400±0.100比1.933±0.153,q=7.239,P<0.01),A1 分化高于 OGD/R 组(2.200±0.200 比 1.600±0.100,q=9.000,P<0.01),A2分化低于 OGD/R组(1.133±0.047 比 1.433±0.058,q=5.510,P<0.05).OGD/R+LA+BAY组A1 分化低于 OGD/R+LA组(0.846±0.050 比 1.000,q=6.527,P<0.01),A2 分化高于 OGD/R+LA 组(1.300±0.100 比 1.000,q=7.491,P<0.05);而 OGD/R+LA+STA 组 A1 分化高于OGD/R+LA组(1.177±0.049 比 1.000,q=7.520,P<0.01),A2 分化低于 OGD/R+LA 组(0.813±0.066 比 1.000,q=4.661,P<0.01).结论 MCT1 调控 SCI 后 AS 乳酸转运,增加 AS 胞内乳酸含量促进其向A2分化,可以通过NF-κB/STAT3信号通路发挥上述调节作用.

Objective To investigate the effect and mechanism ot regulation of lactate transport by monocarboxylate transporter 1(MCT1)on the differentiation of astrocytes(AS)after spinal cord injury(SCI)in rats.Methods Adult female Sprague-Dawley(SD)rats were purchased from the Laboratory An-imal Center of Shanxi Medical University.SCI model was established by modified Allen's method and di-vided into sham operation group,SCI(12 h)group,SCI+LA group and SCI+AZD group with 5 rats in each group.The effects of intraperitoneal injection of exogenous sodium lactate or MCT1 inhibitor on the differentiation of spinal cord AS in rats after SCI were observed.The spinal cord AS of SD rats were cul-tured and the oxygen glucose deprivation/reoxygenation(OGD/R)model was established.First,the lactate content and MCT1 expression of AS treated with OGD/R at different time points were observed.Secondly,the effect of adding exogenous sodium lactate or MCT1 inhibitor after OGD/R on AS differentiation was ob-served,and normal group,OGD/R(OGD4.5 h/R12 h)group,OGD/R+sodium lactate group,OGD/R+sodium lactate+AZD group were set up.Finally,the effects of Bay 11-7082(NF-κB inhibitor)or Stattic(STAT3 inhibitor)pathway proteins on AS differentiation were analyzed,and OGD/R+sodium lactate group,OGD/R+sodium lactate+BAY group,OGD/R+sodium lactate+STA group were set up.One-way analysis of variance was used for statistical analysis,and Tukey's test was used for pairwise com-parison.Results In SCI 12 h group,the expression of spinal cord MCT1 was higher than in sham group(2.776±0.353 vs.1.000,q=13.460,P<0.01).In SCI+LA group,the expression of spinal cord MCT1(2.767±0.208 vs.1.900±0.100,q=10.840,P<0.01)and S100A10 protein(1.933±0.153 vs.1.630±0.061,q=5.924,P<0.05)were higher than in SCI group,and that of C3 was lower than in SCI group(1.603±0.035 vs.1.830±0.147,q=4.561,P<0.05).In SCI+AZD group,the expres-sion of MCT1(1.233±0.153 vs.1.900±0.100,q=8.341,P<0.05)and S100A10 protein(1.237±0.067 vs.1.630±0.061,q=7.681,P<0.01)was lower than in SCI group,and that of C3 was higher than in SCI group(2.340±0.082 vs.1.830±0.147,q=10.260,P<0.01).In the OGD4.5 h/R12 h group,the expression of MCT1 was higher than in normal group(1.760±0.053 vs.1.000,q=6.415,P<0.01).In OGD/R+LA group,the intracellular lactate content of AS(0.028±0.001 vs.0.022±0.001,q=6.415,P<0.01),the expression of MCT1(2.200±0.100 vs.1.633±0.058,q=13.870,P<0.01)and p-STAT3 protein(3.400±0.173 vs.2.133±0.252,q=12.850,P<0.01)were higher than in OGD/R group,the expression of nuclear NF-κB protein was lower than in OGD/R group(1.153±0.129 vs.1.933±0.153,q=12.100,P<0.01),A1 differentiation was lower than in OGD/R group(1.133±0.058 vs.1.600±0.100,q=7.000,P<0.01),and A2 differentiation was higher than in OGD/R group(2.300±0.173 vs.1.433±0.058,q=15.920,P<0.01).In OGD/R+LA+AZD group,the intracellular lactate content of AS(0.014±0.002 vs.0.022±0.001,q=9.278,P<0.01),the expression of MCT1(1.430±0.082 vs.1.633±0.058,q=4.976,P<0.01)and p-STAT3 protein(1.533±0.153 vs.2.133±0.252,q=6.085,P<0.05)were lower than in OGD/R group,nuclear NF-κB protein was higher than in OGD/R group(2.400±0.100 vs.1.933±0.153,q=7.239,P<0.01),A1 differentiation was higher than in OGD/R group(2.200±0.200 vs.1.600±0.100,q=9.000,P<0.01),A2 differentiation was lower than in OGD/R group(1.133±0.047 vs.1.433±0.058,q=5.510,P<0.05).In OGD/R+LA+BAY group,A1 differentiation was lower than in OGD/R+LA group(0.846±0.050 vs.1.000,q=6.527,P<0.01),and A2 differentiation was higher than in OGD/R+LA group(1.300±0.100 vs.1.000,q=7.491,P<0.05).In OGD/R+LA+STA group,A1 differentiation was higher than in OGD/R+LA group(1.177±0.049 vs.1.000,q=7.520,P<0.01),and A2 differentiation was lower than in OGD/R+LA group(0.813±0.066 vs.1.000,q=4.661,P<0.01).Conclusion MCT1 regulates AS lactate transport after SCI,and increased intracellu-lar lactate content in AS can promote the differentiation to A2 of AS,which can be achieved by NF-κB/STAT3 signaling pathway.