桑黄酮G对缺血性中风大鼠血脑屏障的保护作用

Protective effect of Kuwanon G on the blood-brain barrier in ischemic stroke rats

目的探讨桑黄酮G(kuwanon G,KG)对缺血性中风大鼠脑水肿和血脑屏障(BBB)的影响。方法 2018年10月至2019年9月选取45只250~300 g SPF级雄性SD大鼠,采用随机数字表法分为假手术组、缺血性中风模型组(模型组)和治疗组,利用单侧大脑中动脉阻塞(middle cerebral artery occlusion,MCAo)模型,术前3 d假手术组、模型组给予无菌水灌服,治疗组给予KG灌服,术后连续灌服7 d,通过神经功能评分检测各组大鼠的脑损伤程度,利用检测大脑含水量评估各组大鼠脑水肿程度,利用蛋白质印迹法(western blotting)检测各组大鼠脑组织中胞质紧密粘连蛋白-1(zonula occludens-1,ZO-1)、闭合蛋白(occludin)、紧密连接蛋白(claudin-5)和基质金属蛋白-9(matrix metalloproteinase 9,MMP-9)蛋白的表达。结果与假手术组相比[6 h(0.30±0.48)、1 d(0.30±0.48)、3 d(0.30±0.48)、7 d(0.30±0.48),模型组(6 h(15.20±1.14)、1 d(14.60±1.17)、3 d(15.50±0.85)、7 d(15.60±1.08)]神经功能明显受损;治疗组[6 h(14.80±1.03)、1 d(13.20±1.03)、3 d(12.10±1.2)、7 d(9.40±1.27)]神经功能虽受损,但经桑黄酮G治疗后神经功能显著恢复,在第7天尤为显著(P<0.001)。与假手术组[1 d(73.83±0.91)%、2 d(73.89±0.82)%、3 d(73.89±0.82)%、7 d(74.97±0.38)%]相比,模型组[1 d(81.71±1.12)%、2 d(84.98±0.39)%、3 d(83.54±0.84)%、7 d(80.84±1.22)%]脑水肿明显,在术后2天脑水肿最严重;治疗组[1 d(76.85±2.41)%、2 d(77.81±1.69)%、3 d(76.87±1.97)%、7 d(76.55±1.27)%]脑水肿亦明显,但经治疗后脑水肿明显减轻(P<0.001)。相比假手术组ZO-1(0.52±0.15)、Occludin(0.52±0.15)、Claudin-5(0.89±0.06)、MMP-9(0.42±0.02),模型组ZO-1(0.21±0.05)、Occludin(0.38±0.07)、Claudin-5(0.41±0.07)蛋白的表达明显下降,MMP-9(0.86±0.14)显著上升;经桑黄酮G治疗后ZO-1(0.51±0.10)、Occludin(0.89±0.04)、Claudin-5(0.75±0.04)的表达明显上升,而MMP-9(0.28±0.06)的表达显著减少(P<0.001)。结论 KG通过调节MCAo大鼠紧密连接蛋白和基质金属蛋白的表达,保护BBB完整性,从而发挥神经保护作用。

Objective To investigate the effects of kuwanon G(KG)on brain edema and Blood Brain Barrier(BBB)in ischemic stroke rats.Methods Forty-five adult male SD rats(250-300g)were enrolled from October 2018 and September 2019,and were assigned into three groups by random number table method:control group(Sham),middle cerebral artery occlusion(MCAo)and treatment group(KG).Sham and MCAo groups were treated with sterile water for 3 days before operation,and KG group was treated with KG(5 mg/kg)for 3 days before surgery.After MCAo operation all groups were treated 7 days as before.The severity of brain injury in each group was measured by modified neurological severity score.Brain edema was detected by water content.The expression of ZO-1,Occludin,Claudin-5 and MMP-9 in the brain was tested by Western blotting.Results Compared with the Sham group[6 h(0.30±0.48),1 d(0.30±0.483),3 d(0.30±0.48),7 d(0.30±0.48)],MCAo group[6 h(15.20±1.14),1 d(14.60±1.17),3 d(15.50±0.85),7 d(15.60±1.08)]nemurological function was significantly impaired,the neurological function was significantly recovered after treatment with KG[6 h(14.80±1.03),1 d(13.20±1.03),3 d(12.10±1.20),7 d(9.40±1.27)],especially on day 7(P<0.001).The brain water content in MCAo group[1 d(81.71±1.12)%,2 d(84.98±0.39)%,3 d(83.54±0.84)%,7 d(80.84±1.22%)]was significantly higher than that in the sham group[1 d(73.83±0.91)%,2 d(73.89±0.82)%,3 d(73.89±0.82)%,7 d(74.97±0.38%)],especially on day 2 after surgery;the brain water content significantly reduced after treatment with KG[1 d(76.85±2.41)%,2 d(77.81±1.69)%,3 d(76.87±1.97)%,7 d(76.55±1.27%)](P<0.001).Compared the level of ZO-1(0.52±0.15),Occludin(0.52±0.15),Claudin-5(0.89±0.06)and MMP-9(0.42±0.02)in sham group,the expressions of ZO-1(0.21±0.05),Occludin(0.38±0.07)and Claudin-5(0.41±0.07)in the model group were significantly decreased,while MMP-9(0.86±0.14)was significantly increased;The expression of ZO-1(0.51±0.10),Occludin(0.89±0.04)and Claudin-5(0.75±0.04)were significantly increased after treatment with KG,while the expression of MMP-9(0.28±0.06)was significantly decreased(P<0.001).Conclusion Kuwanon G play a neuroprotective role,reduce brain edema and protect the integrity of BBB by regulating the expression of tight junction proteins and matrix metalloprotein in MCAo rats.