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JTradit Chin Med 2014 October 15; 34(5): 576-583 ISSN 0255-2922 © 2014 JTCM. All rights reserved.
EXPERIMENTAL STUDY
Protective effect of Tanreqing injection on axon myelin damage in the brain of mouse model for experimental autoimmune encephalo-myelitis
Yongping Fan, Tao Yang, Qi Zheng, Lei Wang, Chunxiao Yuan, Ling Fang, Kangning Li
Yongping Fan, Tao Yang, Chunxiao Yuan, Kangning Li,
Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China Qi Zheng, Lei Wang, Ling Fang, School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China
Supported by the Research on the Effect of Catalpol on the OPCs' Proliferation and Differentiation (No. 81173237) and Research on the Impact of OPCs and Remyelination via the Method of Bushenyisu with EAE mice (No. 81072765) of National Natural Science Foundation
Correspondence to: Prof. Yongping Fan, Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China. yong-pingf@hotmail.com Telephone: +86-10-67096665 Accepted: January 19,2014
Abstract
OBJECTIVE: To evaluate the effect of Tanreqing injection on axon myelin in the mouse brain of experimental autoimmune encephalomyelitis (EAE).
METHODS: An EAE model was established by myelin oligodendrocyte glycoprotein (MOG) nization in C57BL/6 mice. Mice were randomly divided into the following groups: normal, model, prednisone acetate (PA) (6 mg/kg), Tanreqing high dose (5.14 mL/kg), Tanreqing low dose (2.57 mL/ kg). On the day of immunization, both Tanreqing groups were treated by intraperitoneal injection, with the PA group treated by intragastrical perfusion after T cell response, and the other groups treated with saline. Changes in body weight, neurological deficit score, incidence rate, mortality rate,
and course of disease were observed for all mice. Brain tissue was isolated and stained with hematox-ylin-eosin, and pathological investigations performed to evaluate axon myelin damage by transmission electron microscopy (TEM). Myelin basic protein and microtubule associated protein-2 were analyzed by immunohistochemistry.
RESULTS: Tanreqing injection significantly prolonged EAE latency and decreased the neurological deficit score, alleviated infiltration of inflammatory cells in the focus area, up-regulated hippocampal MBP expression at the acute stage and the remission stage, and increased microtubule associated protein-2 expression in the EAE brain to varying degrees in the acute stage. TEM analysis indicated that Tanreqing injection alleviates myelin damage in the EAE mouse and maintains the integrity of circular layer structures and alleviates axon mitochondrial swelling.
CONCLUSION: Tanreqing injection alleviates EAE symptoms.
©2014 JTCM. All rights reserved.
Key words: Multiple sclerosis; Encephalomyelitis, autoimmune, experimental; Tanreqing injection; Myelin basic protein; Microtubule-associated proteins
INTRODUCTION
Multiple sclerosis (MS) is a chronic inflammatory de-myelinating disease of the central nervous system
(CNS) and is mediated by T cell immunity,1-4 leading to CNS demyelination, and damage to oligodendrocyte axons.5 It is the most commonly seen CNS disease for young people.6 Glucocorticoids and interferon are commonly used to treat MS, but there is no effective way to prevent its recurrence.7-10 Oligodendrocytes are the main CNS gliocyte and function by encircling the axon to form myelin. When MS occurs, oligodendrocytes are attacked by T-cells, inducing myelin damage. Damage to myelin is a key pathogenic mechanism of MS, and how to prevent and treat such damage has become the key to treating MS/experimental autoimmune encephalomyelitis (EAE). Use of multiple myelin oligodendrocyte glycopeptides (MOG) 35-55 to induce EAE in C57BL/6 mice is an important component of modern MS research. Tanreqing is composed of Huangqin (Radix Scutellariae Baicalensis), Ling-yangjiao (Cornu Saigae Tataricae), Jinyinhua (Flos Lonicerae), and Lianqiao (Fructus Forsythiae Suspensae). Clinically, it is used to effectively treat acute cerebral ischemia, infantile acute pneumonia, and acute chole-cystitis.11,12 Animal experiments relieve inflammatory response of pyohemia and resist endotoxin action by decreasing high mobility group box 1 protein (HMGB1)
in the rat.13,14
In the present study, changes in the extent of infiltrating inflammatory cells, myelin basic protein (MBP) expression, and microtubule associated protein-2 (MAP-2) in the brain of EAE mice were observed with HE staining and immuno-histochemical methods. Myelin damage was assessed by transmission electron microscopy (TEM) to probe the effects of Tanreqing injection on myelin during EAE pathological progression.
MATERIALS AND METHODS
Animals
Female specific pathgen free (SPF) grade C57BL/6 mice (n=100, 7-8 weeks of age, weighing 18-22 g), were supplied by Beijing Wei Tong Li Hua Experimental Animals Technique Co., Ltd., (Beijing, China), and raised at the Center of Experimental Animals, Capital Medical University (Beijing, China). All experimental procedures were approved by the Institutional Animal Care Committee of Capital Medical University.
Drugs and reagents
Tanreqing injection was supplied by Shanghai Kaibao Pharmacy Co., Ltd., (Shanghai, China). Prednisone acetate (PA) was supplied by Pacific Pharmacy Co., Ltd., (Tianjin, China). MOG35-55 (MEVGWYRSPFSRVVH-LYRNGK) was synthesized by Kangwei Shiji Bio-Science and Technology Co., Ltd., (Beijing, China). Complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were supplied by Sigma (St. Louis, MO, USA). Tubercusis (H37Ra, TB) was produced by Difco Company (St. Agnes, Corpus Christi, TX, USA). Rabbit an-
ti-MBP and anti-MAP-2 were purchased from Abcam (St. Barry, MA, USA). Rabbit ultra-sensitive two-step kits (PV-9001), concentrating type DAB kits (ZLI-9017/9018/9019) were purchased from Beijing Zhongshan Jinqiao Company (Beijing, China). Xylol, absolute ethanol, paraformaldehyde, chloral hydrate, glutaraldehyde, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and other routine reagents were supplied by Beijing Chemical Reagent Company (Beijing, China).
Preparation of the EAE model
Mice were randomly divided into 4 groups by random number table method: normal, model, PA, Tanreqing high dose, and Tanreqing low dose groups, with 20 mice in each group. 10 mg MOG35-55 and 120 mg TB were dissolved with 20 mL saline, mixed with 20 mL CFA, and repeatedly whipped until formation of an antigen emulsion of oil-wrapping-water shape. The mixed antigen emulsion was injected into four subcutaneous points on both sides of the back middle line on EAE mice. On the day of immunization (day 0) and at 48 h, PTX 500 ng (0.2 mL)/mouse was intra-abdomi-nally injected to induce EAE. Mice in the normal group were injected with saline.
Treatment methods
On the day of immunization, the mice in the Tanreqing high dose and low dose groups were treated by in-tra-abdominal injection (5.14 and 2.57 mL/kg, respectively) for 40 days. The PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response, and other groups were treated by saline (10 mL/kg) for 40 days. The response and changes in body weight and behavior were observed. Additionally, response latency, incidence rate, and mortality were calculated.
Observation of indices
The response latency, incidence rate, and mortality of EAE mice were calculated. Nervous system symptoms were assessed by commonly-used 0-5 grade criteria:15,16 grade 0: normal; grade 1: dystonia of tail; grade 2: partial paralysis of hind limbs or unstable gait; grade 3: complete paralysis of hind limbs; grade 4: partial or complete paralysis of fore limbs; and grade 5: moribund condition or death. On the 21st day (peak symptoms) and 40th day (remission recovery) following immunization, mice were sacrificed, with 10 mice at each time point in each group. From each group, two mice were taken for fixation with glutaraldehyde and observed with TEM after staining. The other mice were fixed with paraformaldehyde, dehydrated, imbedded with paraffin, sectioned, and pathological changes observed after HE staining. Changes in MBP and MAP-2 expression were detected by immunohistochemical methods, and integrated optic density (IOD) was determined with NIS-Elements BR 3.2 software (Nikon, Melville, NY, USA), and statistical analysis performed.
Preparation for histopathologic investigation
Anesthesia (10% chloral hydrate, 0.35 mL/kg) was injected intraperitoneally prior to histology. Blood was evacuated with saline from the right auricle for 5 min and perfusion conducted with paraformaldehyde for 10 min. The brain and spinal cord were separated and placed into paraformaldehyde for epoxy and preservation.
Detection of laboratory indices
For HE staining, the following steps were conducted: incubation in xylol for 2x 15 min and 100% alcohol for 2x5 min, followed by 80% alcohol 5 min, and distilled water for 5 min, before staining with hematoxy-lin solution for 5 min. Samples were then rinsed with hematoxylin solution and running water for 2 s, 1% hydrochloric acid in alcohol for 2 s, distilled water for 20 s, distilled water for 2 s, and then stained with 0.5% eosin for 3 min. Samples were then washed with distilled water for 2 s, 80% alcohol for 2 s, 95% alcohol for 2 s, absolute ethanol 5 min, carbol-xylol for 5 min, xylol for 3x2 min, and then mounted with neutral resin.
IHC staining
Paraffin sections were put into citrate buffer for antigen repair after deparaffinization, rinsed with PBS, cooled at room temperature for 30-40 min, incubated in 3% H2O2 for 10 min, treated with the first antigen, then incubated at 4T! overnight, then placed at room temperature for 1 h. The second antigen was added and incubated at 37T!, and developed with DAB for 1 min. After dehydration and mounting with neutral resin, slides were observed by optical microscopy. IOD was determined with NIS-Elements BR 3.2 software (Nikon, Tokyo, Japan) and statistical analysis performed.
Preparation for electron microscopy
White matter and gray matter tissue were cut into small pieces of 1 mm2, and fixed with 3% glutaralde-hyde for 2 h, washed three times for 10 min with 0.1 phosphate buffer solution (PBS), fixed with 1% osmic acid, and washed three times for 10 min with 0.1 PBS. Slides were then dehydrated with alcohol for 10 min at each step: 100% alcohol 2x15 min; 100% acetone 2x 15 min, and immersed into embedding medium of low viscosity (1: 1) for 4 h. Embedding medium was exposed to air overnight and placed into pure embed-
ding medium for 4 h, and polymerized at 37T! for 24 h. Samples were then stained with uranium acetate for 30 min, rinsed three times with water, stained with lead nitrate for 20 min, and rinsed once with NaOH followed by two water rinses.
Statistical process
The data are expressed as mean ± standard deviation ( x ± s). SPSS 16.0 (Version 16.0, International Business Machines Corporation, Chicago, IL, USA) statistical software was used for data analysis. Normality tests was carried out and ANOVA used for those exhibiting normality, otherwise the rank test was used. P<0.05 was regarded as statistically significant.
RESULTS
Changes in incidence rate, mortality rate, and latency
The incidence rate in the EAE model group was 100%, with the mortality rates in both the model and Tanreqing high dose groups at 7%. Notably, the Tanreqing high dose group latency was prolonged compared with the Tanreqing low dose group (P<0.05) (Table 1).
Changes of nervous function scores
The EAE mice experienced a T cell response on the 8th day following immunization. The mice in the model group were listless, experienced a reduction in body weight or slowed growth, ruffled hair, and exhibited hy-poactivity. After displaying symptoms of an impaired nervous system, the mice showed tail weakness, a staggering gait, hind limb paralysis, locomotion difficulty, paralysis of all four limbs, and even death. EAE mice reached peak symptoms on day 15, and on day 21 the nervous system function score in all treatment groups decreased to some extent. Notably, on days 32 and 40, all the treatment groups were different compared with the model group (P<0.01) (Table 2).
Histopathological changes
HE staining indicated that in the normal brain, structures were in good order, complete, and the cytoplasm and cell nuclei of nerve cells were clear, with scattered neuroglia cells, very few lymphocytes around small veins. In all EAE model groups, infiltration of inflam-
Table 1 The EAE incidence rate, mortality rate, and latency of EAE mice (x ±s)
Incidence rate (%)
Mortality rate (%) Latency (days)
Model PA
Tanreqing high dose Tanreqing low dose
б mg/kg 5.14 mL/kg 2.57 mL/kg
100 100 100 100
ll.2±l.9 10.9±2.2 ll.9±l.9 ll.2±l.9a
Notes: model group was treated by saline (10 mL/kg) for 40 days, PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response, Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). PA: prednisone acetate. Compared with the Tanreqing high dose group, aP<0.05.
matory cells and solidification of nervous cell nuclei around the small blood vessels in the brain could be seen at the acute stage, and both gray and white matter were involved. Inflammation was located mainly around the third cerebral and lateral ventricles. At the acute stage, lymphocytes aggregated around blood vessels and formed a typical sleeve-shape. Lymphocyte infiltration could still be seen during remission, but this was alleviated to some extent compared with that at the acute stage. Pathological changes of the brain in all the treatment groups were relieved to varying degrees (Figure 1).
Changes in the brain observed with TEM
TEM analysis of acute stage EAE indicated that the cir-
Table 2 Changes of nervous system function scores ( x ±s)
cular layer structure of myelin was intact in controls, with neurofilaments and neuromicrotubules exhibiting a natural density and order. Synaptic structure and mitochondrial shape were also normal. However, EAE mice exhibited a circular layer structure and deformed, loose myelin, with separation of lamellar layers showing hair-like changes. Additionally, axons appeared injured, marked by local enlargement and mitochondria lysosomes were swollen and deformed, although ill-defined crista were not obvious, or even lacking altogether. In all treatment groups, the loose deformation of the circular layer structures was alleviated to varying degrees, with milder injury of the axon compared with the model group (Figure 2).
Group n Day 0 Day 10 Day 15 Day 21 Day 32 Day 40
Model 5 0.00±0.00 0.50±0.49 2.13±0.79 2.35±0.78 2.30±0.54 2.20±0.54
PA 5 0.00±0.00 0.57±0.73 2.03±0.88a 1.10±0.22b 0.80±0.22b 0.60±0.22ь
Tanreqing high dose 5 0.00±0.00 0.27±0.50 2.25±0.70 1.64±0.48b 1.43±0.61" 1.43±0.84bd
Tanreqing low dose 5 0.00±0.00 0.83±1.03a 2.77±0.56bc 1.88±0.69d 1.50±0.71ы 1.43±0.77bd
Notes: model group was treated by saline (10 mL/kg) for 40 days, PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response, Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). PA: prednisone acetate. Compared with the model group, aP<0.05, kP<0.01; compared with the PA group, P< 0.01, dP<0.05.
Figure 1 Pathological changes in EAE brain
A1, A2: normal group; B1, B2: model group; C1, C2: PA group; D1, D2: Tanreqing high dose group; E1, E2: Tanreqing low dose group at the acute stage and remission stage, respectively. Model and normal group were treated by saline (10 mL/kg) for 40 days, PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). EAE: experimental autoimmune encephalomyelitis; PA: prednisone acetate.
Figure 2TEM images of changes in EAE brain
A: normal group; B: model group: C: PA group; D: Tanreqing high dose group; E: Tanreqing low dose group at the acute stage. Model and normal groups were treated by saline (10 mL/kg) for 40 days, PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively).TEM: transmission electron microscopy; EAE: experimental autoimmune encephalomyelitis; PA: prednisone acetate.
Immunohistochemical analysis of MBP
There were significant differences in MBP expression in the cortex by day 21 (Table 3). The normal group exhibited elevated expression compared with all other groups (P<0.01). MBP expression in the model hippocampus DG differed compared with the Tanreqing high dose and low dose groups (P<0.01), whereas PA mice showed decreased expression. Hippocampal DG MBP expression in the Tanreqing high dose group was the highest, compared with the other experimental groups (P<0.01, P<0.05).
MBP expression in hippocampal DG and CA1 areas in the Tanreqing high dose low dose groups were elevated
compared with the model group (P<0.01, P<0.05, respectively) (Table 4). There were no significant differences between groups in MBP expression in the hippocampal CA1 area and the lateral ventricle (Table 4, Figure 3).
Immunohistochemical analysis ofMAP-2
On day 21, MAP-2 expression differed between normal and all model groups in the cortex, hippocampal DG and CA1, and lateral ventricle (P<0.01, P<0.05). There were also expression differences between the model group cortex relative to the Tanreqing high and low dose groups (P<0.01, P<0.05). Hippocampal CA1
Table 3 Integrated optic density of brain MBP at day 21 (x ±s)
Cortex
Hippocampus DG area
Hippocampus CA1 area
Lateral ventricle
Normal 5 200±29 67±14 157±54 143±22
Model 5 157±17a 39±13a 102±19c 122±27c
PA 5 156±35a 48±l4ad 123±35d 154±40d
Tanreqing high dose 5 153±11a 76±7be 107±12a 115±31af
Tanreqing low dose 5 159±17a 53±18cb 101±19a 135±34b
Notes: model and normal groups were treated by saline (10 mL/kg) for 40 days; PA group was treated by intragastric perfusion of PA (6 mg/ kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). MBP: myelin basic protein; PA: prednisone acetate. Compared with the normal group, aP<0.01, P< 0.05; compared with the model group, bP<0.01, P<0.05; compared with the PA group, eP<0.01, P<0.05.
|Table 4 Integrated optic density of brain MBP at day 40 (x ±s) |
Group n Cortex Hippocampus DG area Hippocampus CA1 area Lateral ventricle
Normal 5 200±35 67±14 116±62 154±17
Model 5 129±20a 35±13a 86±24 125±8
PA 5 187±36b 42±l4a 95±33 135±46
Tanreqing high dose 5 126±l4a 54±10b 93±19 119±12
Tanreqing low dose 5 126±22a 49±12cd 76±12 132±15
Notes: model and normal groups were treated by saline (10 mL/kg) for 40 days; PA group was treated by intragastric perfusion of PA (6 mg/ kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). MBP: myelin basic protein; PA: prednisone acetate. Compared with the normal group, aP<0.01, P< 0.05; compared with the model group, bP<0.01, P<0.05.
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Figure 3 MBP changes in EAE brain
A1, A2: normal; B1, B2: model; C1, C2: PA; D1, D2: Tanreqing high dose; E1, E2: Tanreqing low dose at the acute stage and on day 40, respectively. Model and normal groups were treated by saline (10 mL/kg) for 40 days; PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). MBP: myelin basic protein; EAE: experimental autoimmune encephalomyelitis; PA: prednisone acetate.
MAP-2 expression of Tanreqing high and low dose groups is higher than the model and PA groups (P< 0.01, P<0.05) (Table 5, Figure 4). We also observed MAP-2 expression differences in the cerebral cortex, hippocampus CA1 and DG, and lateral ventricle during remission on day 40 relative to controls (P<0.01, P<0.05). Interestingly, there were also differences between the PA and model groups in the cortex, hippocampal DG (P<0.01, P< 0.05), and between the Tanreqing high dose group and the PA cortex, hippocampal CA1 and DG, and lateral ventricle (P<0.01) (Table 6, Figure 4).
DISCUSSION
Tanreqing injection is composed of Huangqin (Radix Scutellariae Baicalensis), Lingyangjiao (Cornu Saigae Ta-taricae), Jinyinhua (Flos Lonicerae), and Lianqiao (Fruc-tus Forsythiae Suspensae). Huangqin (Radix Scutellariae Baicalensis) is a monarch drug that clears heat, drys dampness, purges fire, and removes toxic substances. It clears heat of the upper-jiao, but it is mostly used to treat hepatitis, jaundice, renal infection, myocardial ischemia and other diseases.17-19 Lingyangjiao (Cornu Saigae Tataricae) reduce heat and remove toxic sub-
[Table 6 Integrated optic density of MAP-2 in the brain of EAE mice on day 40 (x ±s) 1
Group n Cortex Hippocampus DG area Hippocampus CA1 area Lateral ventricle
Normal 5 140±17 53±10 98±21 110±16
Model 5 68±7a 28±7a 63±11a 77±9a
PA 5 102±15ab 40±10de 80±15d 99±10
Tanreqing high dose 5 75±24ac 43±16de 61±13a 77±21a
Tanreqing low dose 5 69±6ac 50±11e 60±15a 85±19a
Notes: model and normal groups were treated by saline (10 mL/kg) for 40 days; PA group was treated by intragastric perfusion of PA (6 mg/ kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). EAE: experimental autoimmune encephalomyelitis; MAP-2: microtubule associated protein-2; PA: prednisone acetate. Compared with the normal group, aP<0.01, dP<0.05; compared with the model group, bP<0.01, P<0.05; compared with the PA group, cP<0.01.
|Table 5 Integrated optic density of MAP-2 in the brain of EAE mice on day 21 (x ±s) 1
Group n Cortex Hippocampus DG area Hippocampus CA1 area Lateral ventricle
Normal 5 120±20 48±10 72±22 126±28
Model 5 63±13a 22±5a 40±13a 68±20a
PA 5 78±15a 27±8a 48±13a 72±23a
Tanreqing high dose 5 91±27ac 22±9a 59±19bd 62±9a
Tanreqing low dose 5 93±23bd 21±6a 49±17bd 68±8a
Notes: model and normal groups were treated by saline (10 mL/kg) for 40 days; PA group was treated by intragastric perfusion of PA (6 mg/ kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). EAE: experimental autoimmune encephalomyelitis; MAP-2: microtubule associated protein-2; PA: prednisone acetate. Compared with the normal group, aP<0.01, kP<0.05; compared with the model group, cP<0.01, dP<0.05.
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Figure 4 MAP-2 changes in the EAE brain
A1, A2: normal, B1, B2: model, C1, C2: PA, D1, D2: Tanreqing high dose, E1, E2: Tanreqing low dose at the acute stage and remission stage, respectively. Model and normal groups were treated by saline (10 mL/kg) for 40 days; PA group was treated by intragastric perfusion of PA (6 mg/kg) on the 9th day after T cell response; Tanreqing high dose and low dose groups were treated by intra-abdominal injection for 40 days (5.14 and 2.57 mL/kg, respectively). MAP-2: microtubule associated protein-2; EAE: experimental autoimmune encephalomyelitis; PA: prednisone acetate.
stances, resolving phlegm and relieving spasms, and Jinyinhua (Flos Lonicerae), and Lianqiao (Fructus For-sythiae Suspensae) function reduce fever and remove toxins, dispelling stagnated heat. They together function to reduce fever and remove toxicity, suggesting they might be suitable for the initial stage of MS/EAE. Clinically, Tanreqing injection improves action on hypoxae-mia and other pathological states of acute lung injury, inflammatory exudation of the alveolus and microvas-cular injury induced by infection,13,20 and inhibits viral proliferation in cells and strengthens immune system function.21 TCM holds that "the kidney is in charge of bones and produces marrow which above joins in the brain as marrow sea" and that it joins the spinal column and spinal cord. After many years of clinical and experimental studies, understanding the basic patho-genesis of MS continues to progress. These include kidney deficiency and stagnation of phlegm, tonifying kidney, resolving phlegm, and promotion of blood circulation, which might be used as a basic treatment method for MS. However, during the early stages of MS, phlegm coagulation and accumulation of toxic substances are the main pathogenesis. Tanreqing injection reduces fever, resolves phlegm, and removes toxins, acting mainly on phlegm coagulation in the body to treat acute MS/EAE.
In the present experiment, MOG35-55 was used to immunize C57BL/6 mice to establish an EAE model with pathologic symptoms.22,23 Our results indicate that Tanreqing injection alleviates impairment of nervous function at the acute stage and the remission stage (on days 21, 32, and 40), and that PA treatment demonstrated potential therapeutic action. HE staining at the peak stage indicated an elevated number of inflammatory cells around the veins in the brain, which is a primary factor that induces EAE. The extent of infiltrating inflammatory cells at the acute stage and the remission stage was superior in the Tanreqing low dose group compared with the model group.
Many studies indicate that demyelination and axon damage are the main pathological changes of MS/EAE. MBP is a protein expressed by a specific kind of oligo-dendrocyte, Schwann cells, before formation of the myelin sheath. Myelin sheaths commonly form a membranous lamellar structure with phospholipids, ensuring nervous system impulse conduction, which is an important index for assessing demyelination.24,25 Importantly, myelin sheath formation is attacked by T cells in the inflammatory response.26-29 Oral administration of MBP for treatment of EAE has been reported abroad.30,31 In those experiments, expression of hippocampal MBP following Tanreqing treatment was up-regulated at the acute and remission stage. TEM of Tanreqing low dose mice revealed that myelin damage was alleviated, and circular layer structures were kept intact. In the model group, the circular layer structure was loose and deformed, with prominent hair-like changes. This suggests that Tanreqing provides protection for myelin in EAE mice.
MAP-2 is an important cytoskeleton protein, closely correlated with nervous system plasticity, and is an important marker for repairing damaged axons and dendrites.32,33 Such damage occurs mainly in the soma, den-drite, dendritic spine, and the dense postsynaptic area of neurons in the central nervous system, which plays an important role in branching and extending dendrites. MAP-2, as a member of the microtubule-associ-ated protein family, an important component for stabilizing cellular microtubules, plays an important role in neurogenesis, and is an essential prerequisite for the proliferation and differentiation of oligodendrocyte precursors and forming functional axons.34,35 The hippocampus is a center of learning and memory and is easily injured by inflammatory responses of the central nervous system.36 We observed MAP-2 expression in the brain of both Tanreqing treatment groups increased to varying degrees at the acute EAE stage, protecting axons from damage in the cortex and hippocampal CA1 area. TEM results showed that axon mitochondria swelled, with disturbances to energy metabolism in the model group. These changes were alleviated to varying degrees in both treatment groups, indicating that Tan-reqing injection offers protection to axons. In summary, Tanreqing injection alleviates injury of the myelin sheath in the hippocampus and cerebral cortex in EAE mice, providing a partial experimental rationale for treatment of MS.
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