The Protective Effects And Mechanisms Of Preconditioning With Baicalin On The Axon And Neuron In Experimental Autoimmune Encephalomyelitis Rats

Experimental autoimmune encephalomyelitis (EAE) has beenserved as an animal model. It is an immune disease mediated byautoimmune reactive CD4~+ T lymphocyte cell during the course ofonset demyelinating diseases, Loss of axon and apoptosis of neuron playimportant role and are association with neurological disability in EAE orMS. A key player in the regulation of inflammatory gene expression isthe nuclear factor-kB (NF-κB). Several studies also support theconclusion that NF-κB play important roles in the apoptosis of theneuron. It is generally acknowledged that the therapy of glucocor-ticosteroid on EAE is effective, But these kinds of drug have some sideeffects, So the clinical application of glucocorticosteroid is limited insome extent. Our previous studies have confirmed that baicalin coulddecrease the production of cytokine on infectious brain edema and inhibitthe apoptosis of the neuron on hypoxia-ischemic brain damage. The aimof this experiment is to explore that if preconditioning with baicalin canprotect EAE rats through observing the relation among axonal damageand apoptosis of the neuron with NF-κB in EAE rats.Two sections were included in this experiment.Section one: The protective effect of preconditioning with baicalinon the axon in the experimental autoimmune encephalomyelitis rats Objective: To observe the effects of preconditioning with baicalinon axonal damage and neurological disability in EAE rats. Methods:Ninety-six Wistar rats aged six-eight-weeks were used as research subjectand randomly divided into four groups:①Control group(CON, n=24);②Experimental autoimmune encephalomyelitis group(EAE, n=24);③Preconditioning with dexamethasone (DXM,n=24);④Preconditioningwith baicalin (BAC, n=24). On the day of immunization, DXM wasgiven intraperitoneally daily with a dose of 0.5 mg/200g for 1 week ingroup DXM; BAC was given intraperitoneally daily with a dose of 200mg/kg for 1 week in group BAC; normal sodium(NS) was givenintraperitoneally with the same volume for 1 week in groups CON andEAE. neurological score, weight, morbidity were examined daily afterimmunization. The spinal cord and brain stem were removed on day 7th,14th,21th postimmunization, and stained with hematoxylin and eosin.Myelin staining with LOYEZ. Neurofilament-200KD (NF-20KDa) wasdetected by immunohistochemistry in spinal cord. Results:①Manifestation: There is no signs in group CON. The morbidity was87.50％(14/16), 56.25％(9/16) and 50％(8/16) respectively inGroup EAE, Group DXM, and Group BAC, presenting a lower morbidityin the groups of DXM and BAC than EAE. the difference weresignificant (P＜0.01).A increasing trend of the weight was shown in thethe group CON, but the weight in the group EAE decreased before onset one to two days noticeable decreased on the term of morbidity crest-time,and a rise followed recovery. And significant lower than CON on the 7th,14th and 21th day, the difference were significant (P＜0.01).Weightwere no significant difference between the groups EAE and DXM (P＞0.05). Preconditioning with baicalin can improve the weight than that ofgroup EAE (P＜0.01) . The date of accident were 8.75±0.25, 12.8±0.29 and 13.06±0.23 respectively in groups EAE, DXM and BAC. Itwas postponed in Group DXM, and BAC compared with Group EAE (P＜0.01).The neurological scores were 1.56±0.34 and 1.46±0.20respectively in groups DXM and BAC. It was lower in groupsDXM and BAC compared with Group EAE (2.78±0.31), the differencewas significant (P＜0.01).②Pathological changes: The number ofinflammatory foci were 3.00±1.83 and 4.65±1.24 respectively inGroups DXM and BAC, which was lower compared with Group EAE(8.21±3.92), there were significantly difference(P＜0.05).③Detectionof NF-200KDa by immunohistochemistry in spinal cord: Axonal densityin group EAE was very lower than group CON at each time point.Preconditioning with dexamethasone and baicalin can improve the axonaldensity compared with group EAE, the difference were significant(P＜0.01). Conclusion: Preconditioning with baicalin can reduceinfiltration of inflammatory cells, protect axon, improve the Manife-station and decrease the neurological score in EAE rats. Section two: The effects and mechanisms of preconditioning withbaicalin on apoptosis of the neuron in experimental autoimmuneencephalomyelitis ratsObjective: To observe the effects of preconditioning with baicalinon the apoptosis of neuron and expression of protein and mRNA aboutNF-κBp65 in EAE rats, and to explore the mechanism of apotosis of theneuron in the spinal cord. Methods: The groups and correspondingprocondition are as same as first part. RNA was extracted and theexpression of NF-κBp65 mRNA detected by RT-PCR. NF-κBp65was detected by immunohistochemistry and apoptosis of the neuron byTUNEL in spinal cord. Results:①Apoptotic index (AI) of neuron:Apoptosis of neuron was seldom seen in group CON. The AI of neuronin group EAE was the highest among three groups at every observationtime, (23.25±1.82,63.00±4.66 and 31.50±3.63 respectively in thegroup EAE; 16.75±1.28,33.88±1.46 and 22.00±2.45 repectively in thegroup DXM; 15.25±1.67,34.25±3.28 and 21.88±3.09 repectively inthe group BAC), there were different significantly in group DXM andBAC compared with group EAE(P＜0.05).②The expression of NF-κBp65 protein: NF-κBp65 positive ceils were seldom seen in the groupCON, but the ratio of it was more increasingly from 7th day to 14th dayand decreased from 21th day postimmunization in the group EAE(19.13±2.53,50.50±8.07 and 33.50±6.16 respectively). The less ratio were seen in the group DXM and BAC compared with EAE (13.12±1.72,28.55±2.49 and 18.00±3.30 in the group DXM; 14.00±1.51,32.75±3.45 and 17.37±2.44 in the group BAC, respectively). There weresignificant difference (P＜0.05).③Detection to NF-κBp65mRNA inspinal cord: Expression of NF-κBp65mRNA didn't change obviously atevery time point in the group CON (0.55±0.065,0.53±0.083 and 0.54±0.082 respectively ). But it increased significantly in the group EAE, anda significant difference was seen in group DXM or group BAC (0.78±0.062,0.98±0.105 and 0.83±0.071 in group EAE, respectively; 0.67±0.040, 0.84±0.051 and 0.68±0.065 in the group DXM, respectively;0.65±0.038, 0.82±0.103 and 0.70±0.069 in group BAC, respectively).there was significant difference compared with EAE group (P＜0.01)Conclusion: The result indicated that preconditioning with baicalin canobviously reduce apoptosis of neuron and the mechanism was correlationwith suppressing activation of the NF-κBp65.