α-硫辛酸对实验性自身免疫性脑脊髓炎大鼠外周血和中枢MMP-9表达的影响
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摘要
目的:多发性硬化(multiple sclerosis,MS)是一种病因尚未完全明确的中枢神经系统自身免疫性疾病,以中枢神经系统多发性炎性脱髓鞘、神经胶质细胞增生以及不同程度的轴索损害为主要病理特征。目前众多研究表明多发性硬化患者早期就存在着血脑屏障(blood-brain barrier ,BBB)的破坏,并推测其可能是多发性硬化患者发病过程中的一个关键性的环节。基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)通过降解血管内皮细胞中的基质蛋白进而增加微血管内皮的通透性,使T细胞等炎症细胞穿透BBB基底膜,导致BBB破坏。α-硫辛酸(α-Lipoic acid,α-LA)被誉为“万能的抗氧化剂”,是已知天然的抗化氧剂中抗氧化能力最强的一种,它不仅能够通过清除氧自由基、降低单核细胞的迁移能力,同时还能稳定BBB,进而抵抗单核细胞黏附引起的氧化应激损伤。
本实验旨在通过建立自身免疫性脑脊髓炎( experimental autoimmune encephalomyelitis,EAE)大鼠模型,检测EAE大鼠外周血和中枢神经系统(central nervous system,CNS)内MMP-9的表达水平,同时观察α-硫辛酸对EAE大鼠外周血和中枢神经系统内MMP-9表达的影响,进而探讨α-硫辛酸对中枢神经系统损伤可能的保护作用及其机制。
方法:Wistar大鼠共125只,雌性,6-8周龄,体重180～220g ,其中80只随机分为2组即EAE组和CFA组,2个组再分别按时间分为免疫后6天组、8天组、10天组、12天组、14天组、16天组、18天组、20天组8个亚组,每个亚组5只。另有45只随机分为3组即对照-EAE组、低剂量-EAE组(免疫后第6天开始给予腹腔注射α-硫辛酸30mg/kg·d)、高剂量-EAE组(免疫后第6天开始给予腹腔注射α-硫辛酸100mg/kg·d),3个组再分别按时间分为免疫后8天组、12天组、20天组3个亚组,每个亚组5只。每天称量体重和作临床评分;取脑组织视交叉和脊髓腰膨大处行HE染色观察炎性细胞的浸润、免疫组织化学染色检测脑组织和脊髓中MMP-9表达量的变化;酶联免疫吸附实验测定血清中MMP-9的含量。
结果:
1发病情况:(Fig.1、2)
EAE大鼠发病急性期的临床表现呈动态性变化,它们大多数在免疫后的第11天开始出现临床症状,病情多在第14天达到高峰,之后逐渐恢复。
2神经功能评分:(Table 1)
高剂量-EAE组大鼠发病高峰期时神经功能评分显著低于对照EAE组和低剂量-EAE组,具有统计学意义(P<0.05)。
低剂量-EAE组大鼠发病高峰期时神经功能评分也低于对照EAE组,具有统计学意义(P<0.05)。
3组织病理学改变:(Fig.3、4)
EAE组12天组、14天组、16天组、18天组大鼠脊髓和脑组织内均可见不同程度的炎性细胞浸润,尤以14天组(即发病高峰期)大鼠脊髓和脑组织内炎性细胞浸润的程度最为显著,在血管周围出现密集的袖套样炎性细胞浸润。
4免疫组化病理学观察:(Fig. 5～10)(Table 2～5)
4.1 EAE组和CFA组两组比较
EAE组8天组、10天组、12天组、14天组、16天组大鼠脊髓中MMP-9阳性细胞数显著高于同期CFA组,具有统计学意义(P<0.05)。
EAE组10天组、12天组、14天组大鼠脑组织中MMP-9阳性细胞数显著高于同期CFA组,具有统计学意义(P<0.01)。
4.2 EAE组间比较
EAE组10天组与6天组、14天组、18天组、20天组相比较,大鼠脊髓腰膨大处MMP-9阳性细胞数较多,且具有统计学意义(P<0.05)。
EAE组12天组与6天组、8天组、14天组、16天组、18天组、20天组相比较,大鼠脑组织视交叉处MMP-9阳性细胞数较多,且具有统计学意义(P<0.05)。
4.3对照EAE组、低剂量-EAE组、高剂量-EAE组三组比较
低剂量-EAE组12天组、20天组与同期对照EAE组相比较,大鼠脊髓MMP-9阳性细胞数并不具有统计学意义(P>0.05)。
高剂量-EAE组12天组、20天组大鼠脊髓MMP-9阳性细胞数显著低于同期对照EAE组,具有统计学意义(P<0.05)。
高剂量-EAE组12天组、20天组大鼠脊髓处MMP-9阳性细胞数显著低于同期低剂量-EAE组,具有统计学意义(P<0.05)。
低剂量-EAE组12天组与对照EAE组比较,大鼠脑组织MMP-9阳性细胞数并不具有统计学意义(P>0.05)。
高剂量-EAE组12天组大鼠脑组织处MMP-9阳性细胞数显著低于同期对照EAE组,具有统计学意义(P<0.01)。
高剂量-EAE组12天组大鼠脑组织处MMP-9阳性细胞数显著低于同期低剂量-EAE组,具有统计学意义(P<0.05)。
5血清中MMP-9含量比较(Table 6、7)
EAE组12天组与CFA组12天组相比较,EAE组大鼠血清中具有更高的MMP-9含量,且具有统计学意义(P<0.01)。
EAE组12天组与6天组、8天组、10天组、14天组、16天组、18天组、20天组相比较,大鼠血清中MMP-9具有统计学意义(P<0.05)。
低剂量-EAE组各组大鼠血清中MMP-9的含量低于同期对照EAE组,具有统计学意义(P<0.05)。
高剂量-EAE组各组大鼠血清中MMP-9的含量低于同期对照EAE组具有统计学意义(P<0.01)。
结论:
1α-硫辛酸可以降低EAE大鼠的发病率,减低疾病本身对机体的神经系统功能损害。
2α-硫辛酸能够降低血脑屏障通透性的标志物MMP-9的水平,具有稳定血脑屏障的作用,提示其在治疗人类MS方面具有很有希望的应用前景。
3α-硫辛酸在治疗EAE大鼠时具有剂量依赖性,大剂量能更有效的抑制疾病的发展,可指导临床用药。
4 EAE发病急性期时外周血和CNS中的MMP-9水平呈现动态变化,且显示与临床表现具有相关性提示其在疾病发生和进展中可能起到了关键性的作用,并可考虑作为该疾病的潜在生物学标记物。
Objective:Multiple sclerosis is an autoimmune disease of the central nervous system,but its cause is not yet fully clear. The main pathological features is that multiple central nervous system inflammatory demyelination,glial cell proliferation and varying degrees of axonal damage . At present a number of studies have shown that patients with multiple sclerosis exist blood-brain barrier damage at the early stage, and speculate that it may be a critical component of the pathogenesis of multiple sclerosis. Matrix metalloproteinase -9 degradates matrix proteins which is in the vascular endothelial cells,and leading to an increased microvascular endothelial permeability, so that T cells and other inflammatory cells can penetrate through the BBB basement membrane,and finally the BBB is damaged.α-lipoic acid which has been known as the "universal antioxidant" is the most effective natural anti-oxygen agent , it is not only scavenging oxygen free radicals, and reducing the migration ability of the monocyte , but also stabilizing the blood-brain barrier ,and resisting the oxidative stress which is induced by the adhesion of the monocytes.
This study is designed to establish a rat model of autoimmune encephalomyelitis,and detect the expression of the MMP-9 levels in the peripheral blood and the central nervous system, while observing the effect ofα-lipoic acid on the level of MMP-9 expression in the peripheral blood and central nervous system of the EAE rat. Discussing the protective effect ofα-lipoic acid on the injury of the central nervous system and its mechanism.
Methods:125 female Wistar rats ,6-8 weeks old, weighing from 180g to 220g.80 rats are randomly divided into two groups(the EAE group and the CFA group),and each of the two groups are further divided into eight subgroups(the 6-day group,the 8-day group,the 10-day group,the 12-day group,the 14-day group,the 16-day group,the 18-day group,and the 20-day group) by time,and each sub-group contains five rats. The other 45 rats are randomly divided into three group (the control-EAE group,the low-doseα-lipoic acid-EAE group which is given intraperitoneally 30mg/kg·d ALA from 6th day after immunization and the high-doseα-lipoic acid-EAE group which is given intraperitoneally 100mg/kg·d ALA from 6th day after immunization), and each of the three groups are further divided into three subgroups(8-day, 12-day group and 20-day group) by time, and each sub-group contains five rats. Weighing every day and making clinical score. observing the infiltration of inflammatory cells by HE staining,and evaluating the level of MMP-9 expression in the brain tissue and spinal cord by immunohistochemical staining. Assessing the serum level of MMP-9 by enzyme-linked immunosorbent.
Results:
1 The clinical appearance: (Fig.1,2)
The clinical appearance of the EAE rats has show dynamic changes during the acute stage of the disease.Most of them emerge the clinical appearance at the 11th day after immunization,and reach the peak of the disease at the 14th day after immunization,and then decline.
2 Neurological function score: (Table 1)
At the peak of the disease,the neurological function score of the high-doseα-lipoic acid-EAE group was significantly lower than the control EAE group and low-doseα-lipoic acid-EAE group (P <0.05).
Low-doseα-lipoic acid-EAE group is also lower than the control EAE group (P <0.05),which is statistical significance.
3 Histopathological changes: (Fig.3,4)
Different degrees of inflammatory lesions appear in the spinal cord and brain tissues in the EAE group(12-day group, 14-day group, 16-day group,and 18-day group). Especially the 14-day group (at the peak of the disease) is the most significant,which appears the sleeve-like dense inflammatory cell infiltration around the blood vessels.
4 Immunohistochemical pathology observation: (Fig. 5～10)(Table 2～5)
4.1 Comparing the two groups( EAE group and the CFA group )
Compared with the CFA group(8-day group, 10-day group, 12-day group, 14-day group, 16-day group), the MMP-9 positive cells in the spinal cord of the EAE group was statistically significant (P <0.05).
Compared with the corresponding CFA group(10-day group, 12-day group, 14-day group)the MMP-9 positive cells in the brain tissue of the EAE group is statistically significant (P <0.01).
4.2 Comparing between the EAE groups
Compared with the 6-day group, 14-day group, 18-day group, 20-day group, the MMP-9 positive cells in the spinal cord lumbar enlargement of the 10-day group was statistically significant (P <0.05).
Compared with the 6-day group, 8-day group, 14-day group, 16-day group, 18-day group, 20-day group, the MMP-9 positive cells in the brain tissue at the optic chiasm of the 10-day group was statistically significant (P <0.05).
4.3 Comparing the three groups(the control EAE group,the low-doseα-lipoic acid-EAE group,and the high-doseα-lipoic acid-EAE group)
Compared with the control EAE group(12-day group, 20-day group) ,the MMP-9 positive cells in the spinal cord lumbar enlargement of the Low-doseα-lipoic acid-EAE group was not statistically significant (P> 0.05).
Compared with the control EAE group(12-day group, 20-day group) ,the MMP-9 positive cells in the spinal cord lumbar enlargement of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).
Compared with the low-doseα-lipoic acid-EAE group(12-day group, 20-day group), the MMP-9 positive cells in the spinal cord lumbar enlargement of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).
Compared with the control EAE group(12-day group) ,the MMP-9 positive cells in the brain tissue of the Low-doseα-lipoic acid-EAE group was not statistically significant (P> 0.05).
Compared with the control EAE group(12-day group) ,the MMP-9 positive cells in the brain tissue of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.01).
Compared with the low-doseα-lipoic acid-EAE group(12-day group), the MMP-9 positive cells in the brain tissue of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).
5 Comparing the serum level of the MMP-9(Table 6,7)
Compared with the 12-day group of the CFA group, the 12-day group of the EAE group have the higher serum MMP-9 level, and which is statistically significant (P <0.01).
Compared with the 6-day group, 8-day group, 10-day group, 14-day group, 16-day group, 18-day group, 20-day group of the EAE group, the 12-day group of the EAE group have the higher serum MMP-9 level, and which is statistically significant (P < 0.05).
Compared with the control EAE group, the serum level of the MMP-9 of the low-doseα-lipoic acid-EAE group have the higher serum MMP-9 level, and which is statistically significant (P < 0.05).
Compared with the control EAE group, the serum level of the MMP-9 of the high -doseα-lipoic acid-EAE group have the higher serum MMP-9 level, and which is statistically significant(P <0.01).
Conclusion:
1Α-lipoic acid can reduce the incidence of EAE rats ,and also can reduce dysfunction of the nervous system which is resulted by the disease itself.
2Α-lipoic acid can reduce the MMP-9 level which is permeability marker of the blood-brain barrier,and have a role in stabling the blood-brain barrier,which is suggesting that it has a promising application prospects in the treatment of human MS.
3Α-lipoic acid with a dose-dependent manner in the treatment of the EAE rats, when large doses can be more effective in suppressing the development of the disease, and which can guide clinical practice.
4 The levels of the MMP-9 in the peripheral blood and CNS of the EAE rats shows dynamic changes during the acute phase of the disease,and which is relevant to the clinical manifestations.It points that MMP-9 may play a key role in the occurrence and progress of the disease, and may be considered as the Potential biological marker of the disease.
本实验旨在通过建立自身免疫性脑脊髓炎( experimental autoimmune encephalomyelitis,EAE)大鼠模型,检测EAE大鼠外周血和中枢神经系统(central nervous system,CNS)内MMP-9的表达水平,同时观察α-硫辛酸对EAE大鼠外周血和中枢神经系统内MMP-9表达的影响,进而探讨α-硫辛酸对中枢神经系统损伤可能的保护作用及其机制。
方法:Wistar大鼠共125只,雌性,6-8周龄,体重180～220g ,其中80只随机分为2组即EAE组和CFA组,2个组再分别按时间分为免疫后6天组、8天组、10天组、12天组、14天组、16天组、18天组、20天组8个亚组,每个亚组5只。另有45只随机分为3组即对照-EAE组、低剂量-EAE组(免疫后第6天开始给予腹腔注射α-硫辛酸30mg/kg·d)、高剂量-EAE组(免疫后第6天开始给予腹腔注射α-硫辛酸100mg/kg·d),3个组再分别按时间分为免疫后8天组、12天组、20天组3个亚组,每个亚组5只。每天称量体重和作临床评分;取脑组织视交叉和脊髓腰膨大处行HE染色观察炎性细胞的浸润、免疫组织化学染色检测脑组织和脊髓中MMP-9表达量的变化;酶联免疫吸附实验测定血清中MMP-9的含量。
结果:
1发病情况:(Fig.1、2)
EAE大鼠发病急性期的临床表现呈动态性变化,它们大多数在免疫后的第11天开始出现临床症状,病情多在第14天达到高峰,之后逐渐恢复。
2神经功能评分:(Table 1)
高剂量-EAE组大鼠发病高峰期时神经功能评分显著低于对照EAE组和低剂量-EAE组,具有统计学意义(P<0.05)。
低剂量-EAE组大鼠发病高峰期时神经功能评分也低于对照EAE组,具有统计学意义(P<0.05)。
3组织病理学改变:(Fig.3、4)
EAE组12天组、14天组、16天组、18天组大鼠脊髓和脑组织内均可见不同程度的炎性细胞浸润,尤以14天组(即发病高峰期)大鼠脊髓和脑组织内炎性细胞浸润的程度最为显著,在血管周围出现密集的袖套样炎性细胞浸润。
4免疫组化病理学观察:(Fig. 5～10)(Table 2～5)
4.1 EAE组和CFA组两组比较
EAE组8天组、10天组、12天组、14天组、16天组大鼠脊髓中MMP-9阳性细胞数显著高于同期CFA组,具有统计学意义(P<0.05)。
EAE组10天组、12天组、14天组大鼠脑组织中MMP-9阳性细胞数显著高于同期CFA组,具有统计学意义(P<0.01)。
4.2 EAE组间比较
EAE组10天组与6天组、14天组、18天组、20天组相比较,大鼠脊髓腰膨大处MMP-9阳性细胞数较多,且具有统计学意义(P<0.05)。
EAE组12天组与6天组、8天组、14天组、16天组、18天组、20天组相比较,大鼠脑组织视交叉处MMP-9阳性细胞数较多,且具有统计学意义(P<0.05)。
4.3对照EAE组、低剂量-EAE组、高剂量-EAE组三组比较
低剂量-EAE组12天组、20天组与同期对照EAE组相比较,大鼠脊髓MMP-9阳性细胞数并不具有统计学意义(P>0.05)。
高剂量-EAE组12天组、20天组大鼠脊髓MMP-9阳性细胞数显著低于同期对照EAE组,具有统计学意义(P<0.05)。
高剂量-EAE组12天组、20天组大鼠脊髓处MMP-9阳性细胞数显著低于同期低剂量-EAE组,具有统计学意义(P<0.05)。
低剂量-EAE组12天组与对照EAE组比较,大鼠脑组织MMP-9阳性细胞数并不具有统计学意义(P>0.05)。
高剂量-EAE组12天组大鼠脑组织处MMP-9阳性细胞数显著低于同期对照EAE组,具有统计学意义(P<0.01)。
高剂量-EAE组12天组大鼠脑组织处MMP-9阳性细胞数显著低于同期低剂量-EAE组,具有统计学意义(P<0.05)。
5血清中MMP-9含量比较(Table 6、7)
EAE组12天组与CFA组12天组相比较,EAE组大鼠血清中具有更高的MMP-9含量,且具有统计学意义(P<0.01)。
EAE组12天组与6天组、8天组、10天组、14天组、16天组、18天组、20天组相比较,大鼠血清中MMP-9具有统计学意义(P<0.05)。
低剂量-EAE组各组大鼠血清中MMP-9的含量低于同期对照EAE组,具有统计学意义(P<0.05)。
高剂量-EAE组各组大鼠血清中MMP-9的含量低于同期对照EAE组具有统计学意义(P<0.01)。
结论:
1α-硫辛酸可以降低EAE大鼠的发病率,减低疾病本身对机体的神经系统功能损害。
2α-硫辛酸能够降低血脑屏障通透性的标志物MMP-9的水平,具有稳定血脑屏障的作用,提示其在治疗人类MS方面具有很有希望的应用前景。
3α-硫辛酸在治疗EAE大鼠时具有剂量依赖性,大剂量能更有效的抑制疾病的发展,可指导临床用药。
4 EAE发病急性期时外周血和CNS中的MMP-9水平呈现动态变化,且显示与临床表现具有相关性提示其在疾病发生和进展中可能起到了关键性的作用,并可考虑作为该疾病的潜在生物学标记物。
Objective:Multiple sclerosis is an autoimmune disease of the central nervous system,but its cause is not yet fully clear. The main pathological features is that multiple central nervous system inflammatory demyelination,glial cell proliferation and varying degrees of axonal damage . At present a number of studies have shown that patients with multiple sclerosis exist blood-brain barrier damage at the early stage, and speculate that it may be a critical component of the pathogenesis of multiple sclerosis. Matrix metalloproteinase -9 degradates matrix proteins which is in the vascular endothelial cells,and leading to an increased microvascular endothelial permeability, so that T cells and other inflammatory cells can penetrate through the BBB basement membrane,and finally the BBB is damaged.α-lipoic acid which has been known as the "universal antioxidant" is the most effective natural anti-oxygen agent , it is not only scavenging oxygen free radicals, and reducing the migration ability of the monocyte , but also stabilizing the blood-brain barrier ,and resisting the oxidative stress which is induced by the adhesion of the monocytes.
This study is designed to establish a rat model of autoimmune encephalomyelitis,and detect the expression of the MMP-9 levels in the peripheral blood and the central nervous system, while observing the effect ofα-lipoic acid on the level of MMP-9 expression in the peripheral blood and central nervous system of the EAE rat. Discussing the protective effect ofα-lipoic acid on the injury of the central nervous system and its mechanism.
Methods:125 female Wistar rats ,6-8 weeks old, weighing from 180g to 220g.80 rats are randomly divided into two groups(the EAE group and the CFA group),and each of the two groups are further divided into eight subgroups(the 6-day group,the 8-day group,the 10-day group,the 12-day group,the 14-day group,the 16-day group,the 18-day group,and the 20-day group) by time,and each sub-group contains five rats. The other 45 rats are randomly divided into three group (the control-EAE group,the low-doseα-lipoic acid-EAE group which is given intraperitoneally 30mg/kg·d ALA from 6th day after immunization and the high-doseα-lipoic acid-EAE group which is given intraperitoneally 100mg/kg·d ALA from 6th day after immunization), and each of the three groups are further divided into three subgroups(8-day, 12-day group and 20-day group) by time, and each sub-group contains five rats. Weighing every day and making clinical score. observing the infiltration of inflammatory cells by HE staining,and evaluating the level of MMP-9 expression in the brain tissue and spinal cord by immunohistochemical staining. Assessing the serum level of MMP-9 by enzyme-linked immunosorbent.
Results:
1 The clinical appearance: (Fig.1,2)
The clinical appearance of the EAE rats has show dynamic changes during the acute stage of the disease.Most of them emerge the clinical appearance at the 11th day after immunization,and reach the peak of the disease at the 14th day after immunization,and then decline.
2 Neurological function score: (Table 1)
At the peak of the disease,the neurological function score of the high-doseα-lipoic acid-EAE group was significantly lower than the control EAE group and low-doseα-lipoic acid-EAE group (P <0.05).
Low-doseα-lipoic acid-EAE group is also lower than the control EAE group (P <0.05),which is statistical significance.
3 Histopathological changes: (Fig.3,4)
Different degrees of inflammatory lesions appear in the spinal cord and brain tissues in the EAE group(12-day group, 14-day group, 16-day group,and 18-day group). Especially the 14-day group (at the peak of the disease) is the most significant,which appears the sleeve-like dense inflammatory cell infiltration around the blood vessels.
4 Immunohistochemical pathology observation: (Fig. 5～10)(Table 2～5)
4.1 Comparing the two groups( EAE group and the CFA group )
Compared with the CFA group(8-day group, 10-day group, 12-day group, 14-day group, 16-day group), the MMP-9 positive cells in the spinal cord of the EAE group was statistically significant (P <0.05).
Compared with the corresponding CFA group(10-day group, 12-day group, 14-day group)the MMP-9 positive cells in the brain tissue of the EAE group is statistically significant (P <0.01).
4.2 Comparing between the EAE groups
Compared with the 6-day group, 14-day group, 18-day group, 20-day group, the MMP-9 positive cells in the spinal cord lumbar enlargement of the 10-day group was statistically significant (P <0.05).
Compared with the 6-day group, 8-day group, 14-day group, 16-day group, 18-day group, 20-day group, the MMP-9 positive cells in the brain tissue at the optic chiasm of the 10-day group was statistically significant (P <0.05).
4.3 Comparing the three groups(the control EAE group,the low-doseα-lipoic acid-EAE group,and the high-doseα-lipoic acid-EAE group)
Compared with the control EAE group(12-day group, 20-day group) ,the MMP-9 positive cells in the spinal cord lumbar enlargement of the Low-doseα-lipoic acid-EAE group was not statistically significant (P> 0.05).
Compared with the control EAE group(12-day group, 20-day group) ,the MMP-9 positive cells in the spinal cord lumbar enlargement of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).
Compared with the low-doseα-lipoic acid-EAE group(12-day group, 20-day group), the MMP-9 positive cells in the spinal cord lumbar enlargement of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).
Compared with the control EAE group(12-day group) ,the MMP-9 positive cells in the brain tissue of the Low-doseα-lipoic acid-EAE group was not statistically significant (P> 0.05).
Compared with the control EAE group(12-day group) ,the MMP-9 positive cells in the brain tissue of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.01).
Compared with the low-doseα-lipoic acid-EAE group(12-day group), the MMP-9 positive cells in the brain tissue of the high-doseα-lipoic acid-EAE group was statistically significant (P <0.05).
5 Comparing the serum level of the MMP-9(Table 6,7)
Compared with the 12-day group of the CFA group, the 12-day group of the EAE group have the higher serum MMP-9 level, and which is statistically significant (P <0.01).
Compared with the 6-day group, 8-day group, 10-day group, 14-day group, 16-day group, 18-day group, 20-day group of the EAE group, the 12-day group of the EAE group have the higher serum MMP-9 level, and which is statistically significant (P < 0.05).
Compared with the control EAE group, the serum level of the MMP-9 of the low-doseα-lipoic acid-EAE group have the higher serum MMP-9 level, and which is statistically significant (P < 0.05).
Compared with the control EAE group, the serum level of the MMP-9 of the high -doseα-lipoic acid-EAE group have the higher serum MMP-9 level, and which is statistically significant(P <0.01).
Conclusion:
1Α-lipoic acid can reduce the incidence of EAE rats ,and also can reduce dysfunction of the nervous system which is resulted by the disease itself.
2Α-lipoic acid can reduce the MMP-9 level which is permeability marker of the blood-brain barrier,and have a role in stabling the blood-brain barrier,which is suggesting that it has a promising application prospects in the treatment of human MS.
3Α-lipoic acid with a dose-dependent manner in the treatment of the EAE rats, when large doses can be more effective in suppressing the development of the disease, and which can guide clinical practice.
4 The levels of the MMP-9 in the peripheral blood and CNS of the EAE rats shows dynamic changes during the acute phase of the disease,and which is relevant to the clinical manifestations.It points that MMP-9 may play a key role in the occurrence and progress of the disease, and may be considered as the Potential biological marker of the disease.
引文
1 Lucchinetti CF , Rodriguez M . The controversy surrounding the pathogenesis of the multiple sclerosis lesion. Mayo Clin Proc. 1997;72(7):665-678
2 Ewing C,Bernard CC.Insights into the aetiology and pathogenesis of multiple sclerosis. Immunol Cell Biol. 1998;76(1):47-54
3董梅,李春岩.多发性硬化的发病机制.国际免疫学杂志.2006;29(2): 100-104
4董梅,刘瑞春,郭力.实验性变态反应性脑脊髓炎发病初期血脑屏障的形态学变化.脑与神经疾病杂志.2004;12(5):355-357
5 Mun-Bryee S,Rosenberg GA.Gelatinase B modulates seleetive opening of the blood-brain barrier during inflammation.Am J Physiol Regul Integr Comp Physiol.1998;274:R1203-R12ll
6 Kieseier BC,Seifert T,Giovannoni G.Matrix metalloproteinasesin inflammatory demyelination: targets for treatment. Neurology. 1999;53(1):20-25
7 Kouwenhoven M,Ozenci V,Tjernlund A.Monocyte-derived dendritic cells express and secrete matrix-degrading metalloproteinases and their inhibitors and are imbalanced in multiple sclerosis. J Neuroimmunol. 2002;126(1-2):161-171
8 Vin MC,Lee SM,Kalume F.Autoimmunity due to molecular mimicry as a cause of neurological disease.Nal Med,2002;8(5): 509
9 Hagen TM,Liu J,Lykkesfeldt J.Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc Natl Acad Sci U S A. 2002;99(4):1870-1875
10 Kocak G,Karasu C.Elimination of *O(2)(-)/H(2)O(2) by alpha-lipoic acid mediates the recovery of basal EDRF/NO availability and the reversal of superoxide dismutase-induced relaxation in diabetic rat aorta. Diabetes Obes Metab. 2002;4(1):69-74
11 Schreibelt G,Musters RJ,Reijerkerk A.Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity. J Immunol. 2006;177(4):2630-2637
12 Legge KL,Min B,Bell JJ.Coupling of peripheral tolerance to endogenous interleukin 10 promotes effective modulation of myelin-activated T cells and ameliorates experimental allergic encephalomyelitis. J Exp Med. 2000;191(12):2039-2052
13贾建平,《神经病学》,人民卫生出版社,2008,258-264
14苪德源,《临床神经解剖学》,人民卫生出版社,2007,482-491
15 Chakraborti S,Mandal M,Das S.Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem. 2003;253(1-2):269-285
16 Opdenakker G,Van den Steen PE,Van Damme J.Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol. 2001;22(10):571-579.
17 Opdenakker G,Van den Steen PE,Dubois B.Gelatinase B functions as regulator and effector in leukocyte biology. J Leukoc Biol. 2001;69(6):851-859
18 Matache C,Stefanescu M,Dragomir C.Matrix metalloproteinase-9 and its natural inhibitor TIMP-1 expressed or secreted by peripheral blood mononuclear cells from patients with systemic lupus erythematosus. J Autoimmun. 2003;20(4):323-331
19 Ram M,Sherer Y,Shoenfeld Y.Matrix metalloproteinase-9 and autoimmune diseases. J Clin Immunol. 2006;26(4):299-307
20 Leppert D,Waubant E,Galardy R.T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol. 1995;154(9):4379-4389
21 Romanic AM,White RF,Arleth AJ.Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke. 1998;29(5):1020-1030
22 Chandler S,Coates R,Gearing A.Matrix metalloproteinases degrade myelin basic protein. Neurosci Lett. 1995;201(3):223-226
23 Giraudon P, Malcus C, Chalon A. Astrocytes,cells involved in neuro-immune interactions in the central nervous system.J Soc Biol,2003;197(2):103-112
24 Fainardi E, Castellazzi M, Bellini T.Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis.Mult Scler. 2006;12(3):294-301
25 Fainardi E,Castellazzi M,Bellini T.Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis. Mult Scler. 2006;12(3):294-301
26 Comabella M,Rio J,Espejo C.Changes in matrix metalloproteinases and their inhibitors during interferon-beta treatment in multiple sclerosis. Clin Immunol. 2009;130(2):145-150
27 Morikawa T,Yasuno R,Wada H.Do mammalian cells synthesize lipoic acid? Identification of a mouse cDNA encoding a lipoic acid synthase located in mitochondria. FEBS Lett. 2001;498(1):16-21
28 Haramaki N,Han D,Handelman GJ.Cytosolic and mitochondrial systemsfor NADH-and NADPH-dependent reduction of alpha-lipoic acid. Free Radic Biol Med. 1997;22(3):535-542
29 Smith AR,Shenvi SV,Widlansky M.Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr Med Chem. 2004;11(9):1135-1146
30 Kim HS , Kim HJ , Park KG . Alpha-lipoic acid inhibits matrix metalloproteinase-9 expression by inhibiting NF-kappaB transcriptional activity. Exp Mol Med. 2007;39(1):106-113
1 Leppert D,Waubant E,Galardy R.T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol. 1995;154(9):4379-4389
2 Yong VW,Power C,Forsyth P.Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci. 2001;2(7):502-511
3 Leppert D,Waubant E,Burk MR.Interferon beta-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis. Ann Neurol. 1996;40(6):846-852
4 Stuve O,Dooley NP,Uhm JH.Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9. Ann Neurol. 1996;40(6):853-863
5 Stuve O,Chabot S,Jung SS.Chemokine-enhanced migration of human peripheral blood mononuclear cells is antagonized by interferon beta-1b through an effect on matrix metalloproteinase-9. J Neuroimmunol. 1997;80(1-2):38-46
6 Hu W,Metselaar J,Ben LH.PEG minocycline-liposomes ameliorate CNS autoimmune disease. PLoS One. 2009;4(1):e4151
7 Feng JM.Minireview: expression and function of golli protein in immune system. Neurochem Res. 2007;32(2):273-278
8 Folgueras AR,Fueyo A,Garcia Suarez O.Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice. J Biol Chem. 2008;283(14):9465-9474
9 Pitchekian Halabi H,Campagnoni CW,Skinner E.Strain-related differences in the ability of T lymphocytes to recognize proteins encoded by the golli-myelin basic protein gene. J Neuroimmunol. 1996 ;68(1-2):121-129
10 Tranquill LR,Skinner E,Campagnoni C.Human T lymphocytes specific for the immunodominant 83-99 epitope of myelin basic protein: recognition of golli MBP HOG 7. J Neurosci Res. 1996;45(6):820-828.
11 Katsara M,Deraos G,Tselios T.Design of novel cyclic altered peptide ligands of myelin basic protein MBP83-99 that modulate immune responses in SJL/J mice. J Med Chem. 2008;51(13):3971-3978
12 Matsoukas J,Apostolopoulos V,Kalbacher H.Design and synthesis of a novel potent myelin basic protein epitope 87-99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators. J Med Chem. 2005;48(5):1470-1480
13 Tselios T,Apostolopoulos V,Daliani I.Antagonistic effects of human cyclic MBP(87-99) altered peptide ligands in experimental allergic encephalomyelitis and human T-cell proliferation. J Med Chem. 2002;45(2):275-283
14 Folgueras AR,Fueyo A,Garcia Suarez O.Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice. J Biol Chem. 2008;283(14):9465-9474
15 Shiryaev SA,Savinov AY,Cieplak P.Matrix metalloproteinase proteolysis of the myelin basic protein isoforms is a source of immunogenic peptides in autoimmune multiple sclerosis. PLoS One. 2009;4(3):e4952
16 Lee MA, Palace J, Stabler G.Serum gelatinase B, TIMP-1 and TIMP-2 levels in multiple sclerosis. A longitudinal clinical and MRI study. Brain,1999; 122:191–197
17 Lichtinghagen R,Seifert T,Kracke A.Expression of matrixmetalloproteinase-9 and its inhibitors in mononuclear blood cells of patients with multiple sclerosis. J Neuroimmunol. 1999;99(1):19-26
18 Ozenci V,Rinaldi L,Teleshova N.Metalloproteinases and their tissue inhibitors in multiple sclerosis. J Autoimmun. 1999;12(4):297-303
19 Liuzzi GM,Trojano M,Fanelli M.Intrathecal synthesis of matrix metalloproteinase-9 in patients with multiple sclerosis: implication for pathogenesis. Mult Scler. 2002;8(3):222-228
20 Waubant E,Goodkin DE,Gee L.Serum MMP-9 and TIMP-1 levels are related to MRI activity in relapsing multiple sclerosis. Neurology. 1999;53(7):1397-1401
21 Lindberg RL,De Groot CJ,Montagne L.The expression profile of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in lesions and normal appearing white matter of multiple sclerosis. Brain. 2001 ;124(Pt9):1743-1753
22 Kieseier BC, Kiefer R,Clements JM.Matrix metalloproteinase-9 and -7 are regulated in experimental autoimmune encephalomyelitis. Brain,1998; 121: 159–166
23 Benesova Y,Vasku A,Novotna H.Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis. Mult Scler. 2009;15(3):316-322
24 Avolio C,Ruggieri M,Giuliani F.Serum MMP-2 and MMP-9 are elevated in different multiple sclerosis subtypes.J Neuroimmunol. 2003 ;136(1-2):46-53
25 Birkedal-Hansen H,Moore WG,Bodden MK.Matrix metalloproteinases: a review. Crit Rev Oral Biol Med,1993; 4: 197–250
26 Minagar A,Alexander JS,Schwendimann RN.Combination therapy with interferon beta-1a and doxycycline in multiple sclerosis: an open-label trial. Arch Neurol. 2008;65(2):199-204
27 Schmidt J,Sturzebecher S,Toyka KV.Interferon-beta treatment of experimental autoimmune encephalomyelitis leads to rapid nonapoptotic termination of T cell infiltration. J Neurosci Res. 2001;65(1):59-67
28 Waubant E,Goodkin D,Bostrom A.IFNbeta lowers MMP-9/TIMP-1 ratio, which predicts new enhancing lesions in patients with SPMS. Neurology. 2003;60(1):52-57
29 Galboiz Y,Shapiro S,Lahat N.Matrix metalloproteinases and their tissue inhibitors as markers of disease subtype and response to interferon-beta therapy in relapsing and secondary-progressive multiple sclerosis patients. Ann Neurol. 2001;50(4):443-451
30 Yushchenko M,Mader M,Elitok E.Interferon-beta-1 b decreased matrix metalloproteinase-9 serum levels in primary progressive multiple sclerosis. J Neurol. 2003;250(10):1224-1228
31 Brundula V,Rewcastle NB,Metz LM.Targeting leukocyte MMPs and transmigration: minocycline as a potential therapy for multiple sclerosis. Brain. 2002;125(Pt6):1297-1308
32 LU Pei-yuan, SONG Chun-feng, KOH Chang-sung. Suppression of active experimental autoimmune encephalomyelitis in Lewis rats by using compound ONO-4817, a new inhibitor of matrix metalloproteinases.中国神经免疫学和神经病学杂志, 2003;10(3):177-182
33 Goncalves DaSilva A,Yong VW.Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation. Am J Pathol. 2009;174(3):898-909
2 Ewing C,Bernard CC.Insights into the aetiology and pathogenesis of multiple sclerosis. Immunol Cell Biol. 1998;76(1):47-54
3董梅,李春岩.多发性硬化的发病机制.国际免疫学杂志.2006;29(2): 100-104
4董梅,刘瑞春,郭力.实验性变态反应性脑脊髓炎发病初期血脑屏障的形态学变化.脑与神经疾病杂志.2004;12(5):355-357
5 Mun-Bryee S,Rosenberg GA.Gelatinase B modulates seleetive opening of the blood-brain barrier during inflammation.Am J Physiol Regul Integr Comp Physiol.1998;274:R1203-R12ll
6 Kieseier BC,Seifert T,Giovannoni G.Matrix metalloproteinasesin inflammatory demyelination: targets for treatment. Neurology. 1999;53(1):20-25
7 Kouwenhoven M,Ozenci V,Tjernlund A.Monocyte-derived dendritic cells express and secrete matrix-degrading metalloproteinases and their inhibitors and are imbalanced in multiple sclerosis. J Neuroimmunol. 2002;126(1-2):161-171
8 Vin MC,Lee SM,Kalume F.Autoimmunity due to molecular mimicry as a cause of neurological disease.Nal Med,2002;8(5): 509
9 Hagen TM,Liu J,Lykkesfeldt J.Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc Natl Acad Sci U S A. 2002;99(4):1870-1875
10 Kocak G,Karasu C.Elimination of *O(2)(-)/H(2)O(2) by alpha-lipoic acid mediates the recovery of basal EDRF/NO availability and the reversal of superoxide dismutase-induced relaxation in diabetic rat aorta. Diabetes Obes Metab. 2002;4(1):69-74
11 Schreibelt G,Musters RJ,Reijerkerk A.Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity. J Immunol. 2006;177(4):2630-2637
12 Legge KL,Min B,Bell JJ.Coupling of peripheral tolerance to endogenous interleukin 10 promotes effective modulation of myelin-activated T cells and ameliorates experimental allergic encephalomyelitis. J Exp Med. 2000;191(12):2039-2052
13贾建平,《神经病学》,人民卫生出版社,2008,258-264
14苪德源,《临床神经解剖学》,人民卫生出版社,2007,482-491
15 Chakraborti S,Mandal M,Das S.Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem. 2003;253(1-2):269-285
16 Opdenakker G,Van den Steen PE,Van Damme J.Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol. 2001;22(10):571-579.
17 Opdenakker G,Van den Steen PE,Dubois B.Gelatinase B functions as regulator and effector in leukocyte biology. J Leukoc Biol. 2001;69(6):851-859
18 Matache C,Stefanescu M,Dragomir C.Matrix metalloproteinase-9 and its natural inhibitor TIMP-1 expressed or secreted by peripheral blood mononuclear cells from patients with systemic lupus erythematosus. J Autoimmun. 2003;20(4):323-331
19 Ram M,Sherer Y,Shoenfeld Y.Matrix metalloproteinase-9 and autoimmune diseases. J Clin Immunol. 2006;26(4):299-307
20 Leppert D,Waubant E,Galardy R.T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol. 1995;154(9):4379-4389
21 Romanic AM,White RF,Arleth AJ.Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke. 1998;29(5):1020-1030
22 Chandler S,Coates R,Gearing A.Matrix metalloproteinases degrade myelin basic protein. Neurosci Lett. 1995;201(3):223-226
23 Giraudon P, Malcus C, Chalon A. Astrocytes,cells involved in neuro-immune interactions in the central nervous system.J Soc Biol,2003;197(2):103-112
24 Fainardi E, Castellazzi M, Bellini T.Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis.Mult Scler. 2006;12(3):294-301
25 Fainardi E,Castellazzi M,Bellini T.Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis. Mult Scler. 2006;12(3):294-301
26 Comabella M,Rio J,Espejo C.Changes in matrix metalloproteinases and their inhibitors during interferon-beta treatment in multiple sclerosis. Clin Immunol. 2009;130(2):145-150
27 Morikawa T,Yasuno R,Wada H.Do mammalian cells synthesize lipoic acid? Identification of a mouse cDNA encoding a lipoic acid synthase located in mitochondria. FEBS Lett. 2001;498(1):16-21
28 Haramaki N,Han D,Handelman GJ.Cytosolic and mitochondrial systemsfor NADH-and NADPH-dependent reduction of alpha-lipoic acid. Free Radic Biol Med. 1997;22(3):535-542
29 Smith AR,Shenvi SV,Widlansky M.Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr Med Chem. 2004;11(9):1135-1146
30 Kim HS , Kim HJ , Park KG . Alpha-lipoic acid inhibits matrix metalloproteinase-9 expression by inhibiting NF-kappaB transcriptional activity. Exp Mol Med. 2007;39(1):106-113
1 Leppert D,Waubant E,Galardy R.T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol. 1995;154(9):4379-4389
2 Yong VW,Power C,Forsyth P.Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci. 2001;2(7):502-511
3 Leppert D,Waubant E,Burk MR.Interferon beta-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis. Ann Neurol. 1996;40(6):846-852
4 Stuve O,Dooley NP,Uhm JH.Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9. Ann Neurol. 1996;40(6):853-863
5 Stuve O,Chabot S,Jung SS.Chemokine-enhanced migration of human peripheral blood mononuclear cells is antagonized by interferon beta-1b through an effect on matrix metalloproteinase-9. J Neuroimmunol. 1997;80(1-2):38-46
6 Hu W,Metselaar J,Ben LH.PEG minocycline-liposomes ameliorate CNS autoimmune disease. PLoS One. 2009;4(1):e4151
7 Feng JM.Minireview: expression and function of golli protein in immune system. Neurochem Res. 2007;32(2):273-278
8 Folgueras AR,Fueyo A,Garcia Suarez O.Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice. J Biol Chem. 2008;283(14):9465-9474
9 Pitchekian Halabi H,Campagnoni CW,Skinner E.Strain-related differences in the ability of T lymphocytes to recognize proteins encoded by the golli-myelin basic protein gene. J Neuroimmunol. 1996 ;68(1-2):121-129
10 Tranquill LR,Skinner E,Campagnoni C.Human T lymphocytes specific for the immunodominant 83-99 epitope of myelin basic protein: recognition of golli MBP HOG 7. J Neurosci Res. 1996;45(6):820-828.
11 Katsara M,Deraos G,Tselios T.Design of novel cyclic altered peptide ligands of myelin basic protein MBP83-99 that modulate immune responses in SJL/J mice. J Med Chem. 2008;51(13):3971-3978
12 Matsoukas J,Apostolopoulos V,Kalbacher H.Design and synthesis of a novel potent myelin basic protein epitope 87-99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators. J Med Chem. 2005;48(5):1470-1480
13 Tselios T,Apostolopoulos V,Daliani I.Antagonistic effects of human cyclic MBP(87-99) altered peptide ligands in experimental allergic encephalomyelitis and human T-cell proliferation. J Med Chem. 2002;45(2):275-283
14 Folgueras AR,Fueyo A,Garcia Suarez O.Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice. J Biol Chem. 2008;283(14):9465-9474
15 Shiryaev SA,Savinov AY,Cieplak P.Matrix metalloproteinase proteolysis of the myelin basic protein isoforms is a source of immunogenic peptides in autoimmune multiple sclerosis. PLoS One. 2009;4(3):e4952
16 Lee MA, Palace J, Stabler G.Serum gelatinase B, TIMP-1 and TIMP-2 levels in multiple sclerosis. A longitudinal clinical and MRI study. Brain,1999; 122:191–197
17 Lichtinghagen R,Seifert T,Kracke A.Expression of matrixmetalloproteinase-9 and its inhibitors in mononuclear blood cells of patients with multiple sclerosis. J Neuroimmunol. 1999;99(1):19-26
18 Ozenci V,Rinaldi L,Teleshova N.Metalloproteinases and their tissue inhibitors in multiple sclerosis. J Autoimmun. 1999;12(4):297-303
19 Liuzzi GM,Trojano M,Fanelli M.Intrathecal synthesis of matrix metalloproteinase-9 in patients with multiple sclerosis: implication for pathogenesis. Mult Scler. 2002;8(3):222-228
20 Waubant E,Goodkin DE,Gee L.Serum MMP-9 and TIMP-1 levels are related to MRI activity in relapsing multiple sclerosis. Neurology. 1999;53(7):1397-1401
21 Lindberg RL,De Groot CJ,Montagne L.The expression profile of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in lesions and normal appearing white matter of multiple sclerosis. Brain. 2001 ;124(Pt9):1743-1753
22 Kieseier BC, Kiefer R,Clements JM.Matrix metalloproteinase-9 and -7 are regulated in experimental autoimmune encephalomyelitis. Brain,1998; 121: 159–166
23 Benesova Y,Vasku A,Novotna H.Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis. Mult Scler. 2009;15(3):316-322
24 Avolio C,Ruggieri M,Giuliani F.Serum MMP-2 and MMP-9 are elevated in different multiple sclerosis subtypes.J Neuroimmunol. 2003 ;136(1-2):46-53
25 Birkedal-Hansen H,Moore WG,Bodden MK.Matrix metalloproteinases: a review. Crit Rev Oral Biol Med,1993; 4: 197–250
26 Minagar A,Alexander JS,Schwendimann RN.Combination therapy with interferon beta-1a and doxycycline in multiple sclerosis: an open-label trial. Arch Neurol. 2008;65(2):199-204
27 Schmidt J,Sturzebecher S,Toyka KV.Interferon-beta treatment of experimental autoimmune encephalomyelitis leads to rapid nonapoptotic termination of T cell infiltration. J Neurosci Res. 2001;65(1):59-67
28 Waubant E,Goodkin D,Bostrom A.IFNbeta lowers MMP-9/TIMP-1 ratio, which predicts new enhancing lesions in patients with SPMS. Neurology. 2003;60(1):52-57
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