木犀草素对实验性脑缺血大鼠的脑保护作用及机制研究
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摘要
脑血管病是我国居民死亡的第一原因和成人残疾的第一原因，以缺血性脑血管病最为常见，具有发病率高、病死率高、致残率高、复发率高、恢复缓慢的特点。缺血后继发性脑损伤是患者的病情加重和影响预后的重要原因。近年来，脑缺血后脑损伤的病理生理机制研究已取得了重大进步，然而，临床治疗效果却非常有限。
脑梗死后的继发性脑损伤常导致病情加重，主要包括氧化应激、炎症反应、凋亡、细胞内钙超载、细胞毒性作用、活性氧及自由基等，这几种因素之间相互作用、相互影响，构成复杂的调控网络，形成恶性循环，造成一系列病理级联反应，其中过度炎症反应、氧化应激和凋亡存在于脑梗死后坏死和缺血区域，成为导致继发损伤的主要原因。因此，抗氧化应激、减轻炎症损伤、抑制凋亡成为治疗脑梗死的主要途径之一。
木犀草素（Luteolin）是一种天然黄酮类化合物，分子式为：C15H10O6，存在于多种植物中，因最初是从木犀草科（Resedaceae）木犀草属草本植物木犀草（ResedaodorataL）的叶、茎、枝中分离出而得名，具有多种药理活性，如抗炎、抗氧化、抑制凋亡、抗过敏、抗肿瘤、抗菌、抗病毒、增强免疫功能等多种生物学作用，其毒副作用低，具有良好的临床应用潜力。
本研究选用雄性Sprague-Dawley大鼠，应用改良Longa线栓法建立大鼠右侧大脑中动脉阻塞（middle cerebral artery occlusion, MCAO）模型。在大鼠永久性大脑中动脉阻塞所致的脑缺血模型上观察脑缺血后TLR4，TLR5，P38MAPK，ERK1/2，claudin-5，Bcl-2和Bax的表达，大脑皮层梗塞灶超氧化物歧化酶（Superoxide Dismutase，SOD）与过氧化氢酶（Catalase，CAT）活性及丙二醛（Malondiadehyde，MDA）的含量。确定缺血后与炎症反应、氧化损伤及凋亡的关系，并选用黄酮类化合物木犀草素进行干预，评价干预前后梗死体积、脑含水量、神经功能缺损评分，以及木犀草素对TLR4，TLR5，P38MAPK，ERK1/2，claudin-5，Bcl-2和Bax的表达信号通路的影响，研究木犀草素对缺血损伤后的脑保护作用机制进行初步探讨，并检测木犀草素对氧化应激标志物SOD，CAT活性和MDA含量的影响。本研究分三部分，现将各部分内容概述如下。
第一部分木犀草素对实验性脑缺血大鼠的抗氧化作用及机制研究
目的：观察实验性大鼠脑缺血损伤后不同时间点梗死体积、脑含水量、神经功能缺损评分，claudin-5的表达变化以及SOD，CAT的活性和MDA的含量。研究木犀草素对脑缺血大鼠的脑保护作用。探讨木犀草素抗氧化作用的相关机制。
方法：选用成年健康雄性Sprague-Dawley大鼠为研究对象，应用改良Longa线栓法制备大鼠右侧大脑中动脉梗塞模型（MCAO）。实验1：观察木犀草素的神经保护作用，梗塞后24h和72h分别观察神经功能缺损评分，脑含水量，梗塞体积。实验动物随机分为6组，假手术组（Sham），假手术后腹腔注射等量生理盐水+1%DMSO；手术组（MCAO），缺血术后腹腔注射等量生理盐水；溶剂对照组（MC+DM），缺血术后腹腔注射等量生理盐水+1%DMSO；木犀草素小剂量组（Luteolin-L），缺血术后腹腔注射木犀草素5mg/kg；木犀草素中剂量组（Luteolin-M），缺血术后腹腔注射木犀草素10mg/kg；木犀草素大剂量组（Luteolin-H），缺血术后腹腔注射木犀草素25mg/kg。实验2：观察木犀草素的抗氧化功能。检测脑组织中超氧化物歧化酶（SOD），过氧化氢酶（CAT）的活性，丙二醛（MDA）含量的变化情况。实验动物随机分为5组，假手术组（Sham），手术组（MCAO），溶剂对照组（MC+DM），木犀草素中剂量组（Luteolin-M），木犀草素大剂量组（Luteolin-H）。实验3：观察脑缺血后木犀草素对血脑屏障的影响。实验动物随机分为5组，假手术组（Sham），手术组（MCAO），溶剂对照组（MC+DM），木犀草素中剂量组（Luteolin-M），木犀草素大剂量组（Luteolin-H）。各组取材前进行神经功能评分，干湿重法测定脑组织含水量，TTC染色评价脑梗死体积，分别用RT-qPCR的方法和Westernblot的方法检测缺血脑组织claudin-5mRNA和蛋白水平的变化。
结果：
1Sham组无神经功能缺损，神经功能缺损评分为0分；MCAO组和MC+DM组在24h和72h神经功能缺损评分均较严重，Luteolin-L组在24h和72h与MCAO组和MC+DM组相比没有明显降低神经功能缺损，差异无统计学意义（P＞0.05），Luteolin-M组和Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低神经功能缺损，差异有统计学意义（P＜0.05）。
2Luteolin-L组在24h和72h与MCAO组和MC+DM组相比均没有明显降低脑含水量，差异无统计学意义（P＞0.05），Luteolin-M组和Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低脑含水量，差异有统计学意义（P＜0.05）。
3Luteolin-L组在24h和72h与MCAO组和MC+DM组相比均没有明显降低脑梗死体积，差异无统计学意义（P＞0.05），Luteolin-M组和Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低脑梗死体积，差异有统计学意义（P＜0.05）。我们可以看到木犀草素在每天10mg/kg（Luteolin-M）和25mg/kg（Luteolin-H）时发挥更好的治疗作用，因此，下面的研究集中在10mg/kg和25mg/kg。
4Western blot中，与MCAO组和MC+DM组比较，Luteolin-H组在24h和72h均可明显增加claudin-5的蛋白表达，差异有统计学意义（P＜0.05）。Luteolin-M组只在72h可明显增加claudin-5的蛋白表达，差异有统计学意义（P＜0.05）。
与Western blot的结果一致，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增加claudin-5的mRNA表达，差异有统计学意义（P＜0.05）。Luteolin-M组只在72h可明显增加claudin-5的mRNA表达，差异有统计学意义（P＜0.05）。
5与MCAO组和MC+DM组比较，Luteolin-M组和Luteolin-H组在24h和72h均可明显增加SOD，CAT的活性，差异有统计学意义（P＜0.05）。与MCAO组和MC+DM组比较，Luteolin-M组和Luteolin-H组在24h和72h均可明显减低MDA的含量，差异有统计学意义（P＜0.05）。
结论：木犀草素对局灶性脑缺血大鼠有较好的神经保护作用，可改善脑缺血损伤后的神经功能缺失，减轻脑含水量，减小脑梗死体积，诱导claudin-5的表达，增加SOD，CAT的活性，减少MDA的含量，因此我们推测木犀草素的神经保护作用可能是通过增加claudin-5的表达，提高SOD，CAT的活性，减少MDA含量实现的；这些是木犀草素发挥其抗氧化作用之一，从而实现其神经保护作用。
第二部分木犀草素对实验性脑缺血大鼠的抗炎作用及机制研究
目的：观察脑缺血后不同时间点TLRs/MAPKs/NF-κB的表达变化，研究木犀草素对脑缺血大鼠的抗炎作用，探讨木犀草素抗炎作用的相关信号机制。
方法：选用成年健康雄性Sprague-Dawley大鼠为研究对象，应用改良Longa线栓法制备大鼠右侧大脑中动脉梗塞模型（MCAO）。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水+1%DMSO；手术组（MCAO），缺血术后腹腔注射等量生理盐水；溶剂对照组（MC+DM），缺血术后腹腔注射等量生理盐水+1%DMSO；木犀草素中剂量组（Luteolin-M），缺血术后腹腔注射木犀草素10mg/kg；木犀草素大剂量组（Luteolin-H），缺血术后腹腔注射木犀草素25mg/kg。各组取材前进行神经功能评分，分别用免疫组化，Western-blot和RT-qPCR的方法检测缺血脑组织中TLR4、TLR5、p-p38，p-ERK，NF-κB蛋白和mRNA水平的变化。
结果：
1缺血后24h和72h，TLR4、TLR5、p-p38、NF-κB的表达均有升高，而p-ERK的表达在24h时呈逐渐升高趋势，72h后逐渐降低。两种剂量木犀草素干预后均可以降低脑缺血大鼠的炎症表达。免疫组化中，与MCAO组和MC+DM组相比，Luteolin-H组在24h和72h均可明显降低TLR4、TLR5、p-p38、NF-κB的细胞数，增加p-ERK的细胞数。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显降低TLR4、TLR5、p-p38、NF-κB的细胞数，增加p-ERK的细胞数，差异有统计学意义（P＜0.05）。
2Western blot中，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低TLR4、TLR5、p-p38、NF-κB的蛋白表达，增加p-ERK的蛋白表达。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显降低TLR4、TLR5、p-p38、NF-κB的蛋白表达，增加p-ERK的蛋白表达，差异有统计学意义（P＜0.05）。
3与免疫组化和Western blot的结果一致，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低TLR4、TLR5、p-p38、NF-κB的mRNA表达，增加p-ERK的mRNA表达，差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显降低TLR4、TLR5、p-p38、NF-κB的mRNA表达，增加p-ERK的mRNA表达，差异有统计学意义（P＜0.05）。
结论：局灶性脑缺血后，缺血脑组织的TLR4、TLR5、p-p38、p-ERK、NF-κB表达的变化过程与脑组织损伤程度一致，提示TLR4/NF-κB通路的炎症损伤参与了脑缺血损伤；木犀草素可下调TLR4、TLR5、p-p38、抑制NF-κB的核转位，上调p-ERK，从而减轻缺血脑组织的过度炎症反应，实现缺血后的神经保护作用。
第三部分木犀草素对实验性脑缺血大鼠的抗凋亡作用及机制研究
目的：观察脑缺血损伤后不同时间点Bcl-2/Bax的表达变化，研究木犀草素对脑缺血大鼠的抗凋亡作用，探讨木犀草素抗凋亡作用的相关信号转导机制。
方法：选用成年健康雄性Sprague-Dawley大鼠为研究对象，应用改良Longa线栓法制备大鼠右侧大脑中动脉梗塞模型（MCAO）。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水+1%DMSO；手术组（MCAO），缺血术后腹腔注射等量生理盐水；溶剂对照组（MC+DM），缺血术后腹腔注射等量生理盐水+1%DMSO；木犀草素中剂量组（Luteolin-M），缺血术后腹腔注射木犀草素10mg/kg；木犀草素大剂量组（Luteolin-H），缺血术后腹腔注射木犀草素25mg/kg。各组取材前进行神经功能评分，分别用免疫组化，Western-blot和RT-qPCR的方法检测缺血脑组织中Bcl-2和Bax蛋白和mRNA水平的变化。
结果：
1缺血后24h和72h，Bcl-2的表达降低，Bax表达升高，免疫组化中，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增高Bcl-2的细胞数，降低Bax的细胞数。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显增高Bcl-2的细胞数，降低Bax的细胞数。差异有统计学意义（P＜0.05）。
2Western blot中，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增加Bcl-2的蛋白表达，降低Bax的蛋白表达。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显增加Bcl-2的蛋白表达，降低Bax的蛋白表达。差异有统计学意义（P＜0.05）。
3与免疫组化和Western blot的结果一致，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增加Bcl-2的mRNA表达水平，降低Bax的mRNA表达水平。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显增加Bcl-2的mRNA表达水平，降低Bax的mRNA表达水平。差异有统计学意义（P＜0.05）。
结论：局灶性脑缺血后，缺血脑组织的Bcl-2与Bax表达的变化过程与脑组织损伤程度一致，提示Bcl-2与Bax通路参与了脑缺血损伤；木犀草素可上调Bcl-2的表达并下调Bax的表达，从而减轻缺血脑组织的过度凋亡过程，实现缺血后的神经保护作用。
Cerebral ischemia is the most common type of cerebrovascular disease,which is the first leading cause of death and the most frequent cause ofpermanent disability in adults. Ischemic stroke is one of the main diseasesendangering people’s health in our state, which has a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.Brain tissue injuries secondary to ischemia aggravated the illness and inhibitedthe recovery of patients. Despite advances in the understanding of thepathophysiology of cerebral ischemia in recent years, therapeutic optionsremain limited.
Brain tissue injuries secondary to ischemia often leads to an aggravatedillness. The pathological mechanisms that trigger ischemic brain damage couldbe related to oxidative stress, inflammation, apoptosis, overproduction ofreactive oxygen species, intracellular acidosis, increased release of excitatoryamion acids, destabilization of intracellular calcium and so on. Thesepathophysiologic processes overlap and intercommunicate then form a viciouscycle which results in a series of pathological cascade. Among them excessiveinflammation, oxidative stress and apoptosis are the main causes of thesecondary injury. Therefore, antioxidant stress, reduce inflammation damage,inhibiting apoptosis treatment become one of the main ways for cerebralinfarction.
Luteolin (3′,4′,5,7-tetrahydroxyflavone), an important member of theflavonoid family, is present in various fruits and vegetables. Its molecularformula for: C15H10O6. It exhibits a wide spectrum of pharmacologicalproperties including anti-inflammatory, anti-oxidation, inhibiting apoptosis,anti-allergy, anti-tumor, anti-bacterial, anti-viral and enhancing immunefunction and so on. Besides, it has been proven to have little side effects, andwith good potential clinical application.
Male, healthy Sprague-Dawley rats were used and subjected to modifiedpermanent middle cerebral artery occlusion (pMCAO), as described by Longapreviously. The expression of TLR4, TLR5, P38MAPK, ERK1/2, claudin-5,Bcl-2and Bax were detected in the pMCAO; activites of superoxidedismutase (SOD) and catalase (CAT), and malondiadehyde (MDA) contentwere measured in the ipsilateral cortex. Luteolin’s neuroprotective effect wasanalyzed. Neurological deficits, brain water content and infarct volume wereevaluated. The expression of TLR4, TLR5, P38MAPK, ERK1/2, claudin-5,Bcl-2and Bax were detected; activites of superoxide dismutase (SOD) andcatalase (CAT), and malondiadehyde (MDA) content were measured.
PartⅠAnti-oxidative effect of luteolin and the underlying mechanism inrat experimental stroke
Objective: The aim of this study was to evaluate the neuroprotectiveeffects of luteolin and the underlying mechanisms in cerebral ischemia. Brainwater content, infarct volume, the expression of claudin-5, activites ofsuperoxide dismutase (SOD) and catalase (CAT) and malondiadehyde (MDA)content were measured.
Methods: Male, healthy Sprague-Dawley rats were used and subjected tomodified permanent middle cerebral artery occlusion (pMCAO). Experiment1was used to analyze luteolin’s neuroprotective effect. Neurological deficits,brain water content and infarct volume were evaluated at24h and72h afterpMCAO. Rats were randomly assigned to six groups: Sham-vehicle group(Sham): animals received sham operation and equal volume of DMSO;MCAO group (MCAO): animals received MCAO and equal volume of NaCl0.9%saline; MCAO-vehicle group (MC+DM): animals received MCAO andequal volume of DMSO; and luteolin groups: animals received MCAO andtreated with low dose of luteolin5mg/kg (Luteolin-L), middle dose of luteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H).Luteolin was administered immediately after MCAO and then continued with daily injections for2days. Experiment2was used to analyze luteolin’santi-oxidative effect. SOD, CAT activities, and MDA content were measuredat24h and72h after pMCAO. Rats were randomly assigned to five groups:Sham-vehicle group (Sham): animals received sham opera-tion and equalvolume of DMSO; MCAO group (MCAO): animals received MCAO andequal volume of NaCl0.9%; MCAO-vehicle group (MC+DM): animalsreceived MCAO and equal volume of DMSO; and luteolin groups: animalsreceived MCAO and treated with middle dose of luteolin10mg/kg(Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H). Luteolin wasadministered immediately after MCAO and then continued with dailyinjections for2days. Experiment3was used to detect luteolin's influence onblood-brain barrier (BBB). Rats in this part were assigned to five groups as thesame way of experiment2. Western blotting and RT-qPCR were used to detectthe expression of claudin-5.
Results:
1Rats in Sham group had no palsy and a neurological score of zero. Ratsin MCAO group, MC+DM group, low dose group, middle dose group andhigh dose group performed a left palsy. Compared with MCAO group andMC+DM group, there was a significant improvement in neurological functionscores in the Luteolin-H group and Luteolin-M group both at24h and72h (P<0.05). By contrast, there was no significant effect in Luteolin-L groupcompared with MCAO and MC+DM groups both at24h and72h (P>0.05for all).
2Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group reduced the brain water content significantly both at24hand72h (P <0.05). Luteolin-L group had no effect on brain water contentboth at24h and72h (P>0.05).
3Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group reduced the infarct volume significantly both at24h and72h (P <0.05). Luteolin-L group had no effect on infarct volume both at24hand72h (P>0.05). Based on the results above, we demonstrated that luteolin delivered at high dose (25mg/kg) and middle dose (10mg/kg) per day have abetter therapeutic effect after stroke, and therefore we focused on the luteolintreatment at high dose (Luteolin-H) and middle dose (Luteolin-M) per day forsubsequent biochemical and molecular analysis.
4Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group significantly increased the activities of SOD and CAT bothat24h and72h (P <0.05), Luteolin-H and Luteolin-M group significantlydecreased the levels of MDA (P <0.05).
5Compared with MCAO group and MC+DM group, Luteolin-H groupsignificantly up-regulated the expression of claudin-5both at24h and72h inprotein levels (P <0.05), but only at72h in Luteolin-M group (P <0.05). Inagreement with the results of western blotting, the mRNA expression ofclaudin-5was up-regulated in Luteolin-H group compared with MCAO groupand MC+DM group both at24h and72h (P <0.05), but only at72h inLuteolin-M group (P <0.05).
Conclusions: Systemic administration of luteolin is effective which canameliorate the neurological deficit, improve the brain edema, decrease theinfarct size and up-regulate the expression of claudin-5. Luteolin significantlyincreased the activities of SOD, CAT, decreased the levels of MDA. Theunderlying mechanism of this neuroprotection may be involved in increasingthe activities of SOD and CAT, decreasing the levels of MDA andup-regulating claudin-5expression.
PartⅡ Anti-inflammatory effect of luteolin and the underlying
mechanism in rat experimental stroke
Objective: This study is to evaluate the expression ofTLRs/MAPKs/NF-κB in experimental rats after ischemic injury at differenttime points. The aim of this etudy is to explore anti-inflammatory effect ofluteolin and the underlying mechanism in rat experimental stroke.
Methods: Male, healthy Sprague-Dawley rats were used and subjected to modified permanent middle cerebral artery occlusion (pMCAO). Rats wererandomly assigned to five groups: Sham-vehicle group (Sham): animalsreceived sham operation and equal volume of DMSO; MCAO group (MCAO):animals received MCAO and equal volume of NaCl0.9%; MCAO-vehiclegroup (MC+DM): animals received MCAO and equal volume of DMSO; andluteolin groups: animals received MCAO and treated with middle dose ofluteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg(Luteolin-H). Luteolin was administered immediately after MCAO and thencontinued with daily injections for2days. Immunohistochemistry, Westernblotting and RT-qPCR were used to analyse the expression of TLR4, TLR5,NF-κB, p-p38and p-ERK.
Results:
1In immunohistochemistry, compared with Sham group, TLR4, TLR5,p-p38, NF-κB were upregulated at protein level in ischemic brain, but theexpression of phospho-ERK1/2was significantly increased only at24h andthen decreased at72h (P <0.05).Compared with MCAO and MC+DM groups,luteolin in high dose group dramatically decreased the positive cells of TLR4,TLR5, p-p38, NF-κB and increased the positive cells of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).
2In western blotting, compared with MCAO and MC+DM groups,luteolin in high dose group dramatically decreased the protein level of TLR4,TLR5, p-p38, NF-κB and increased the protein level of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).
3In agreement with the results of immunohistochemistry and westernblotting, compared with MCAO and MC+DM groups, luteolin in high dosegroup dramatically decreased the the mRNA expression of TLR4, TLR5,p-p38, NF-κB and increased the mRNA expression of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).
Conclusions: Systemic administration of luteolin is effective which candecrease the expression of TLR4, TLR5, p-p38, NF-κB and increase theexpression of p-ERK. Therefore, the excessive inflammation of the brainischemia was alleviated.
PartⅢ Anti-apoptosis effect of luteolin and the underlying mechanism inrat experimental stroke
Objective: This study is to evaluate the expression of Bcl-2/Bax inexperimental rats after ischemic injury at different time points. The aim of thisstudy is to explore anti-apoptosis effect of luteolin and the underlyingmechanism in rat experimental ischemic stroke.
Methods: Male, healthy Sprague-Dawley rats were used and subjected tomodified permanent middle cerebral artery occlusion (pMCAO). Rats wererandomly assigned to five groups: Sham-vehicle group (Sham): animalsreceived sham opera-tion and equal volume of DMSO; MCAO group(MCAO): animals received MCAO and equal volume of NaCl0.9%;MCAO-vehicle group (MC+DM): animals received MCAO and equal volumeof DMSO; and luteolin groups: animals received MCAO and treated withmiddle dose of luteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H). Luteolin was administered immediately after MCAO andthen continued with daily injections for2days. Immunohistochemistry,Western blotting and RT-qPCR were used to analyse the expression of Bcl-2and Bax.
Results:
1In immunohistochemistry, compared with Sham group, the expressionof Bcl-2was down-regulated at protein level in ischemic brain, but theexpression of Bax was significantly increased (P <0.05).Compared withMCAO and MC+DM groups, luteolin in high dose group dramaticallyincreased the positive cells of Bcl-2and decreased the positive cells of Bax inthe ischemic cortex both at24h and72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).
2In western blotting, compared with MCAO and MC+DM groups,luteolin in high dose group dramatically increased the protein level of Bcl-2and decreased the protein level of Bax in the ischemic cortex both at24h and72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).
3In agreement with the results of immunohistochemistry and westernblotting, compared with MCAO and MC+DM groups, luteolin in high dosegroup dramatically increased the the mRNA expression of Bcl-2anddecreased the mRNA expression of Bax in the ischemic cortex both at24hand72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).
Conclusions: The expression of Bcl-2was down-regulated afterischemia, but the expression of Bax was up-regulated after ischemia. Systemicadministration of luteolin is effective which can increase the expression ofBcl-2and decrease the expression of Bax. Therefore, the excessive apoptosisof the brain ischemia was alleviated.
脑梗死后的继发性脑损伤常导致病情加重，主要包括氧化应激、炎症反应、凋亡、细胞内钙超载、细胞毒性作用、活性氧及自由基等，这几种因素之间相互作用、相互影响，构成复杂的调控网络，形成恶性循环，造成一系列病理级联反应，其中过度炎症反应、氧化应激和凋亡存在于脑梗死后坏死和缺血区域，成为导致继发损伤的主要原因。因此，抗氧化应激、减轻炎症损伤、抑制凋亡成为治疗脑梗死的主要途径之一。
木犀草素（Luteolin）是一种天然黄酮类化合物，分子式为：C15H10O6，存在于多种植物中，因最初是从木犀草科（Resedaceae）木犀草属草本植物木犀草（ResedaodorataL）的叶、茎、枝中分离出而得名，具有多种药理活性，如抗炎、抗氧化、抑制凋亡、抗过敏、抗肿瘤、抗菌、抗病毒、增强免疫功能等多种生物学作用，其毒副作用低，具有良好的临床应用潜力。
本研究选用雄性Sprague-Dawley大鼠，应用改良Longa线栓法建立大鼠右侧大脑中动脉阻塞（middle cerebral artery occlusion, MCAO）模型。在大鼠永久性大脑中动脉阻塞所致的脑缺血模型上观察脑缺血后TLR4，TLR5，P38MAPK，ERK1/2，claudin-5，Bcl-2和Bax的表达，大脑皮层梗塞灶超氧化物歧化酶（Superoxide Dismutase，SOD）与过氧化氢酶（Catalase，CAT）活性及丙二醛（Malondiadehyde，MDA）的含量。确定缺血后与炎症反应、氧化损伤及凋亡的关系，并选用黄酮类化合物木犀草素进行干预，评价干预前后梗死体积、脑含水量、神经功能缺损评分，以及木犀草素对TLR4，TLR5，P38MAPK，ERK1/2，claudin-5，Bcl-2和Bax的表达信号通路的影响，研究木犀草素对缺血损伤后的脑保护作用机制进行初步探讨，并检测木犀草素对氧化应激标志物SOD，CAT活性和MDA含量的影响。本研究分三部分，现将各部分内容概述如下。
第一部分木犀草素对实验性脑缺血大鼠的抗氧化作用及机制研究
目的：观察实验性大鼠脑缺血损伤后不同时间点梗死体积、脑含水量、神经功能缺损评分，claudin-5的表达变化以及SOD，CAT的活性和MDA的含量。研究木犀草素对脑缺血大鼠的脑保护作用。探讨木犀草素抗氧化作用的相关机制。
方法：选用成年健康雄性Sprague-Dawley大鼠为研究对象，应用改良Longa线栓法制备大鼠右侧大脑中动脉梗塞模型（MCAO）。实验1：观察木犀草素的神经保护作用，梗塞后24h和72h分别观察神经功能缺损评分，脑含水量，梗塞体积。实验动物随机分为6组，假手术组（Sham），假手术后腹腔注射等量生理盐水+1%DMSO；手术组（MCAO），缺血术后腹腔注射等量生理盐水；溶剂对照组（MC+DM），缺血术后腹腔注射等量生理盐水+1%DMSO；木犀草素小剂量组（Luteolin-L），缺血术后腹腔注射木犀草素5mg/kg；木犀草素中剂量组（Luteolin-M），缺血术后腹腔注射木犀草素10mg/kg；木犀草素大剂量组（Luteolin-H），缺血术后腹腔注射木犀草素25mg/kg。实验2：观察木犀草素的抗氧化功能。检测脑组织中超氧化物歧化酶（SOD），过氧化氢酶（CAT）的活性，丙二醛（MDA）含量的变化情况。实验动物随机分为5组，假手术组（Sham），手术组（MCAO），溶剂对照组（MC+DM），木犀草素中剂量组（Luteolin-M），木犀草素大剂量组（Luteolin-H）。实验3：观察脑缺血后木犀草素对血脑屏障的影响。实验动物随机分为5组，假手术组（Sham），手术组（MCAO），溶剂对照组（MC+DM），木犀草素中剂量组（Luteolin-M），木犀草素大剂量组（Luteolin-H）。各组取材前进行神经功能评分，干湿重法测定脑组织含水量，TTC染色评价脑梗死体积，分别用RT-qPCR的方法和Westernblot的方法检测缺血脑组织claudin-5mRNA和蛋白水平的变化。
结果：
1Sham组无神经功能缺损，神经功能缺损评分为0分；MCAO组和MC+DM组在24h和72h神经功能缺损评分均较严重，Luteolin-L组在24h和72h与MCAO组和MC+DM组相比没有明显降低神经功能缺损，差异无统计学意义（P＞0.05），Luteolin-M组和Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低神经功能缺损，差异有统计学意义（P＜0.05）。
2Luteolin-L组在24h和72h与MCAO组和MC+DM组相比均没有明显降低脑含水量，差异无统计学意义（P＞0.05），Luteolin-M组和Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低脑含水量，差异有统计学意义（P＜0.05）。
3Luteolin-L组在24h和72h与MCAO组和MC+DM组相比均没有明显降低脑梗死体积，差异无统计学意义（P＞0.05），Luteolin-M组和Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低脑梗死体积，差异有统计学意义（P＜0.05）。我们可以看到木犀草素在每天10mg/kg（Luteolin-M）和25mg/kg（Luteolin-H）时发挥更好的治疗作用，因此，下面的研究集中在10mg/kg和25mg/kg。
4Western blot中，与MCAO组和MC+DM组比较，Luteolin-H组在24h和72h均可明显增加claudin-5的蛋白表达，差异有统计学意义（P＜0.05）。Luteolin-M组只在72h可明显增加claudin-5的蛋白表达，差异有统计学意义（P＜0.05）。
与Western blot的结果一致，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增加claudin-5的mRNA表达，差异有统计学意义（P＜0.05）。Luteolin-M组只在72h可明显增加claudin-5的mRNA表达，差异有统计学意义（P＜0.05）。
5与MCAO组和MC+DM组比较，Luteolin-M组和Luteolin-H组在24h和72h均可明显增加SOD，CAT的活性，差异有统计学意义（P＜0.05）。与MCAO组和MC+DM组比较，Luteolin-M组和Luteolin-H组在24h和72h均可明显减低MDA的含量，差异有统计学意义（P＜0.05）。
结论：木犀草素对局灶性脑缺血大鼠有较好的神经保护作用，可改善脑缺血损伤后的神经功能缺失，减轻脑含水量，减小脑梗死体积，诱导claudin-5的表达，增加SOD，CAT的活性，减少MDA的含量，因此我们推测木犀草素的神经保护作用可能是通过增加claudin-5的表达，提高SOD，CAT的活性，减少MDA含量实现的；这些是木犀草素发挥其抗氧化作用之一，从而实现其神经保护作用。
第二部分木犀草素对实验性脑缺血大鼠的抗炎作用及机制研究
目的：观察脑缺血后不同时间点TLRs/MAPKs/NF-κB的表达变化，研究木犀草素对脑缺血大鼠的抗炎作用，探讨木犀草素抗炎作用的相关信号机制。
方法：选用成年健康雄性Sprague-Dawley大鼠为研究对象，应用改良Longa线栓法制备大鼠右侧大脑中动脉梗塞模型（MCAO）。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水+1%DMSO；手术组（MCAO），缺血术后腹腔注射等量生理盐水；溶剂对照组（MC+DM），缺血术后腹腔注射等量生理盐水+1%DMSO；木犀草素中剂量组（Luteolin-M），缺血术后腹腔注射木犀草素10mg/kg；木犀草素大剂量组（Luteolin-H），缺血术后腹腔注射木犀草素25mg/kg。各组取材前进行神经功能评分，分别用免疫组化，Western-blot和RT-qPCR的方法检测缺血脑组织中TLR4、TLR5、p-p38，p-ERK，NF-κB蛋白和mRNA水平的变化。
结果：
1缺血后24h和72h，TLR4、TLR5、p-p38、NF-κB的表达均有升高，而p-ERK的表达在24h时呈逐渐升高趋势，72h后逐渐降低。两种剂量木犀草素干预后均可以降低脑缺血大鼠的炎症表达。免疫组化中，与MCAO组和MC+DM组相比，Luteolin-H组在24h和72h均可明显降低TLR4、TLR5、p-p38、NF-κB的细胞数，增加p-ERK的细胞数。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显降低TLR4、TLR5、p-p38、NF-κB的细胞数，增加p-ERK的细胞数，差异有统计学意义（P＜0.05）。
2Western blot中，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低TLR4、TLR5、p-p38、NF-κB的蛋白表达，增加p-ERK的蛋白表达。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显降低TLR4、TLR5、p-p38、NF-κB的蛋白表达，增加p-ERK的蛋白表达，差异有统计学意义（P＜0.05）。
3与免疫组化和Western blot的结果一致，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显降低TLR4、TLR5、p-p38、NF-κB的mRNA表达，增加p-ERK的mRNA表达，差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显降低TLR4、TLR5、p-p38、NF-κB的mRNA表达，增加p-ERK的mRNA表达，差异有统计学意义（P＜0.05）。
结论：局灶性脑缺血后，缺血脑组织的TLR4、TLR5、p-p38、p-ERK、NF-κB表达的变化过程与脑组织损伤程度一致，提示TLR4/NF-κB通路的炎症损伤参与了脑缺血损伤；木犀草素可下调TLR4、TLR5、p-p38、抑制NF-κB的核转位，上调p-ERK，从而减轻缺血脑组织的过度炎症反应，实现缺血后的神经保护作用。
第三部分木犀草素对实验性脑缺血大鼠的抗凋亡作用及机制研究
目的：观察脑缺血损伤后不同时间点Bcl-2/Bax的表达变化，研究木犀草素对脑缺血大鼠的抗凋亡作用，探讨木犀草素抗凋亡作用的相关信号转导机制。
方法：选用成年健康雄性Sprague-Dawley大鼠为研究对象，应用改良Longa线栓法制备大鼠右侧大脑中动脉梗塞模型（MCAO）。实验动物随机分为5组，假手术组（Sham），假手术后腹腔注射等量生理盐水+1%DMSO；手术组（MCAO），缺血术后腹腔注射等量生理盐水；溶剂对照组（MC+DM），缺血术后腹腔注射等量生理盐水+1%DMSO；木犀草素中剂量组（Luteolin-M），缺血术后腹腔注射木犀草素10mg/kg；木犀草素大剂量组（Luteolin-H），缺血术后腹腔注射木犀草素25mg/kg。各组取材前进行神经功能评分，分别用免疫组化，Western-blot和RT-qPCR的方法检测缺血脑组织中Bcl-2和Bax蛋白和mRNA水平的变化。
结果：
1缺血后24h和72h，Bcl-2的表达降低，Bax表达升高，免疫组化中，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增高Bcl-2的细胞数，降低Bax的细胞数。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显增高Bcl-2的细胞数，降低Bax的细胞数。差异有统计学意义（P＜0.05）。
2Western blot中，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增加Bcl-2的蛋白表达，降低Bax的蛋白表达。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显增加Bcl-2的蛋白表达，降低Bax的蛋白表达。差异有统计学意义（P＜0.05）。
3与免疫组化和Western blot的结果一致，Luteolin-H组在24h和72h与MCAO组和MC+DM组相比均可明显增加Bcl-2的mRNA表达水平，降低Bax的mRNA表达水平。差异有统计学意义（P＜0.05）。Luteolin-M组只在72h与MCAO组和MC+DM组相比可明显增加Bcl-2的mRNA表达水平，降低Bax的mRNA表达水平。差异有统计学意义（P＜0.05）。
结论：局灶性脑缺血后，缺血脑组织的Bcl-2与Bax表达的变化过程与脑组织损伤程度一致，提示Bcl-2与Bax通路参与了脑缺血损伤；木犀草素可上调Bcl-2的表达并下调Bax的表达，从而减轻缺血脑组织的过度凋亡过程，实现缺血后的神经保护作用。
Cerebral ischemia is the most common type of cerebrovascular disease,which is the first leading cause of death and the most frequent cause ofpermanent disability in adults. Ischemic stroke is one of the main diseasesendangering people’s health in our state, which has a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.Brain tissue injuries secondary to ischemia aggravated the illness and inhibitedthe recovery of patients. Despite advances in the understanding of thepathophysiology of cerebral ischemia in recent years, therapeutic optionsremain limited.
Brain tissue injuries secondary to ischemia often leads to an aggravatedillness. The pathological mechanisms that trigger ischemic brain damage couldbe related to oxidative stress, inflammation, apoptosis, overproduction ofreactive oxygen species, intracellular acidosis, increased release of excitatoryamion acids, destabilization of intracellular calcium and so on. Thesepathophysiologic processes overlap and intercommunicate then form a viciouscycle which results in a series of pathological cascade. Among them excessiveinflammation, oxidative stress and apoptosis are the main causes of thesecondary injury. Therefore, antioxidant stress, reduce inflammation damage,inhibiting apoptosis treatment become one of the main ways for cerebralinfarction.
Luteolin (3′,4′,5,7-tetrahydroxyflavone), an important member of theflavonoid family, is present in various fruits and vegetables. Its molecularformula for: C15H10O6. It exhibits a wide spectrum of pharmacologicalproperties including anti-inflammatory, anti-oxidation, inhibiting apoptosis,anti-allergy, anti-tumor, anti-bacterial, anti-viral and enhancing immunefunction and so on. Besides, it has been proven to have little side effects, andwith good potential clinical application.
Male, healthy Sprague-Dawley rats were used and subjected to modifiedpermanent middle cerebral artery occlusion (pMCAO), as described by Longapreviously. The expression of TLR4, TLR5, P38MAPK, ERK1/2, claudin-5,Bcl-2and Bax were detected in the pMCAO; activites of superoxidedismutase (SOD) and catalase (CAT), and malondiadehyde (MDA) contentwere measured in the ipsilateral cortex. Luteolin’s neuroprotective effect wasanalyzed. Neurological deficits, brain water content and infarct volume wereevaluated. The expression of TLR4, TLR5, P38MAPK, ERK1/2, claudin-5,Bcl-2and Bax were detected; activites of superoxide dismutase (SOD) andcatalase (CAT), and malondiadehyde (MDA) content were measured.
PartⅠAnti-oxidative effect of luteolin and the underlying mechanism inrat experimental stroke
Objective: The aim of this study was to evaluate the neuroprotectiveeffects of luteolin and the underlying mechanisms in cerebral ischemia. Brainwater content, infarct volume, the expression of claudin-5, activites ofsuperoxide dismutase (SOD) and catalase (CAT) and malondiadehyde (MDA)content were measured.
Methods: Male, healthy Sprague-Dawley rats were used and subjected tomodified permanent middle cerebral artery occlusion (pMCAO). Experiment1was used to analyze luteolin’s neuroprotective effect. Neurological deficits,brain water content and infarct volume were evaluated at24h and72h afterpMCAO. Rats were randomly assigned to six groups: Sham-vehicle group(Sham): animals received sham operation and equal volume of DMSO;MCAO group (MCAO): animals received MCAO and equal volume of NaCl0.9%saline; MCAO-vehicle group (MC+DM): animals received MCAO andequal volume of DMSO; and luteolin groups: animals received MCAO andtreated with low dose of luteolin5mg/kg (Luteolin-L), middle dose of luteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H).Luteolin was administered immediately after MCAO and then continued with daily injections for2days. Experiment2was used to analyze luteolin’santi-oxidative effect. SOD, CAT activities, and MDA content were measuredat24h and72h after pMCAO. Rats were randomly assigned to five groups:Sham-vehicle group (Sham): animals received sham opera-tion and equalvolume of DMSO; MCAO group (MCAO): animals received MCAO andequal volume of NaCl0.9%; MCAO-vehicle group (MC+DM): animalsreceived MCAO and equal volume of DMSO; and luteolin groups: animalsreceived MCAO and treated with middle dose of luteolin10mg/kg(Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H). Luteolin wasadministered immediately after MCAO and then continued with dailyinjections for2days. Experiment3was used to detect luteolin's influence onblood-brain barrier (BBB). Rats in this part were assigned to five groups as thesame way of experiment2. Western blotting and RT-qPCR were used to detectthe expression of claudin-5.
Results:
1Rats in Sham group had no palsy and a neurological score of zero. Ratsin MCAO group, MC+DM group, low dose group, middle dose group andhigh dose group performed a left palsy. Compared with MCAO group andMC+DM group, there was a significant improvement in neurological functionscores in the Luteolin-H group and Luteolin-M group both at24h and72h (P<0.05). By contrast, there was no significant effect in Luteolin-L groupcompared with MCAO and MC+DM groups both at24h and72h (P>0.05for all).
2Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group reduced the brain water content significantly both at24hand72h (P <0.05). Luteolin-L group had no effect on brain water contentboth at24h and72h (P>0.05).
3Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group reduced the infarct volume significantly both at24h and72h (P <0.05). Luteolin-L group had no effect on infarct volume both at24hand72h (P>0.05). Based on the results above, we demonstrated that luteolin delivered at high dose (25mg/kg) and middle dose (10mg/kg) per day have abetter therapeutic effect after stroke, and therefore we focused on the luteolintreatment at high dose (Luteolin-H) and middle dose (Luteolin-M) per day forsubsequent biochemical and molecular analysis.
4Compared with MCAO group and MC+DM group, Luteolin-H andLuteolin-M group significantly increased the activities of SOD and CAT bothat24h and72h (P <0.05), Luteolin-H and Luteolin-M group significantlydecreased the levels of MDA (P <0.05).
5Compared with MCAO group and MC+DM group, Luteolin-H groupsignificantly up-regulated the expression of claudin-5both at24h and72h inprotein levels (P <0.05), but only at72h in Luteolin-M group (P <0.05). Inagreement with the results of western blotting, the mRNA expression ofclaudin-5was up-regulated in Luteolin-H group compared with MCAO groupand MC+DM group both at24h and72h (P <0.05), but only at72h inLuteolin-M group (P <0.05).
Conclusions: Systemic administration of luteolin is effective which canameliorate the neurological deficit, improve the brain edema, decrease theinfarct size and up-regulate the expression of claudin-5. Luteolin significantlyincreased the activities of SOD, CAT, decreased the levels of MDA. Theunderlying mechanism of this neuroprotection may be involved in increasingthe activities of SOD and CAT, decreasing the levels of MDA andup-regulating claudin-5expression.
PartⅡ Anti-inflammatory effect of luteolin and the underlying
mechanism in rat experimental stroke
Objective: This study is to evaluate the expression ofTLRs/MAPKs/NF-κB in experimental rats after ischemic injury at differenttime points. The aim of this etudy is to explore anti-inflammatory effect ofluteolin and the underlying mechanism in rat experimental stroke.
Methods: Male, healthy Sprague-Dawley rats were used and subjected to modified permanent middle cerebral artery occlusion (pMCAO). Rats wererandomly assigned to five groups: Sham-vehicle group (Sham): animalsreceived sham operation and equal volume of DMSO; MCAO group (MCAO):animals received MCAO and equal volume of NaCl0.9%; MCAO-vehiclegroup (MC+DM): animals received MCAO and equal volume of DMSO; andluteolin groups: animals received MCAO and treated with middle dose ofluteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg(Luteolin-H). Luteolin was administered immediately after MCAO and thencontinued with daily injections for2days. Immunohistochemistry, Westernblotting and RT-qPCR were used to analyse the expression of TLR4, TLR5,NF-κB, p-p38and p-ERK.
Results:
1In immunohistochemistry, compared with Sham group, TLR4, TLR5,p-p38, NF-κB were upregulated at protein level in ischemic brain, but theexpression of phospho-ERK1/2was significantly increased only at24h andthen decreased at72h (P <0.05).Compared with MCAO and MC+DM groups,luteolin in high dose group dramatically decreased the positive cells of TLR4,TLR5, p-p38, NF-κB and increased the positive cells of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).
2In western blotting, compared with MCAO and MC+DM groups,luteolin in high dose group dramatically decreased the protein level of TLR4,TLR5, p-p38, NF-κB and increased the protein level of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).
3In agreement with the results of immunohistochemistry and westernblotting, compared with MCAO and MC+DM groups, luteolin in high dosegroup dramatically decreased the the mRNA expression of TLR4, TLR5,p-p38, NF-κB and increased the mRNA expression of p-ERK1/2in theischemic cortex at all time points (P <0.05), while middle dose group onlyshowed effect at72h (P <0.05).
Conclusions: Systemic administration of luteolin is effective which candecrease the expression of TLR4, TLR5, p-p38, NF-κB and increase theexpression of p-ERK. Therefore, the excessive inflammation of the brainischemia was alleviated.
PartⅢ Anti-apoptosis effect of luteolin and the underlying mechanism inrat experimental stroke
Objective: This study is to evaluate the expression of Bcl-2/Bax inexperimental rats after ischemic injury at different time points. The aim of thisstudy is to explore anti-apoptosis effect of luteolin and the underlyingmechanism in rat experimental ischemic stroke.
Methods: Male, healthy Sprague-Dawley rats were used and subjected tomodified permanent middle cerebral artery occlusion (pMCAO). Rats wererandomly assigned to five groups: Sham-vehicle group (Sham): animalsreceived sham opera-tion and equal volume of DMSO; MCAO group(MCAO): animals received MCAO and equal volume of NaCl0.9%;MCAO-vehicle group (MC+DM): animals received MCAO and equal volumeof DMSO; and luteolin groups: animals received MCAO and treated withmiddle dose of luteolin10mg/kg (Luteolin-M) and high dose of luteolin25mg/kg (Luteolin-H). Luteolin was administered immediately after MCAO andthen continued with daily injections for2days. Immunohistochemistry,Western blotting and RT-qPCR were used to analyse the expression of Bcl-2and Bax.
Results:
1In immunohistochemistry, compared with Sham group, the expressionof Bcl-2was down-regulated at protein level in ischemic brain, but theexpression of Bax was significantly increased (P <0.05).Compared withMCAO and MC+DM groups, luteolin in high dose group dramaticallyincreased the positive cells of Bcl-2and decreased the positive cells of Bax inthe ischemic cortex both at24h and72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).
2In western blotting, compared with MCAO and MC+DM groups,luteolin in high dose group dramatically increased the protein level of Bcl-2and decreased the protein level of Bax in the ischemic cortex both at24h and72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).
3In agreement with the results of immunohistochemistry and westernblotting, compared with MCAO and MC+DM groups, luteolin in high dosegroup dramatically increased the the mRNA expression of Bcl-2anddecreased the mRNA expression of Bax in the ischemic cortex both at24hand72h (P <0.05), while middle dose group only showed effect at72h (P <0.05).
Conclusions: The expression of Bcl-2was down-regulated afterischemia, but the expression of Bax was up-regulated after ischemia. Systemicadministration of luteolin is effective which can increase the expression ofBcl-2and decrease the expression of Bax. Therefore, the excessive apoptosisof the brain ischemia was alleviated.
引文
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