Therapeutic Effects Of Traditional Chinese Medicine Xuezhikang On Experimental Autoimmune Neuritis

Background and objectives:Acute inflammatory demyelinating polyneuropathy (AIDP), is a common type of the Guillain-Barre Syndrome (GBS), which is characterized as an autoimmune disease mediated by CD4+T cells by its demyelination in peripheral nerves system (PNS). It is universally accepted that Experimental autoimmune neuritis (EAN), as a valid animal model of GBS, showing similar pathogenesis, pathomechanism, and clinical manifestation to AIDP. EAN induced by heterogeneous peripheral nerve myelin or its components P2 or PO peptides is characterized by a single spontaneously resolving episode of neuritis. It is not only cellular but humoral immunity are involved in its pathogenesis, and we still have a lot to do.Xuezhikang, extract of red yeast rice, a traditional Chinese medicine widely used as lipid-lowering drugs, containing natural lovastatin and its homologues. Red Yeast Rice (RYR) is a traditional Chinese food that is fermented and obtained after red yeast (Monascus purpureus) is grown on rice. It is thought Monacolin K, (also known as Lovastatin), as well as a family of 8 other Monacolin-related substances, played the role of cholesterol-lowering effect in RYR. Of course, the most important action of RYR is thought to be mimicking the statins. However, besides the HMG-CoA reductase inhibitor, there are many other substances also play a part in lowering cholesterol, for example, sterols, isoflavones, and monounsaturated fatty acids. Statins,3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, involved in the mevalonate pathway for cholesterol biosynthesis, has turned into the primary pharmacological pathway to lowering levels of low-density lipoprotein cholesterol (LDL-C). There are many clinical trials had indicated that statins had potentials in reducing coronary events and decreasing coronary heart disease mortality. Previous research in our team had proved that atorvastatin could alleviated EAMG, which was associated with up-regulating the expression of Foxp3 in thymus and the secretion of IL-4 in lymph nodes, as well as a decrease of anti-R97-116 peptide (region 97-116 of the rat acetylcholine receptor a subunit) in serum. However, the problems of tolerance and safety limited the use of statins. The most commonly known is statin-associated myalgia, and the recurrence of myalgia can be still high when patients are challenged with a second statin. Whether Xuezhikang exerts the similar function to statin and what is the effect of Xuezhikang on GBS/EAN? So far, there has been no study about it. In this study, we investigated the effect of Xuezhikang on EAN and attempted to explore the mechanisms. We found that Xuezhikang and Statins have both similarities and differences.Our data indicated the suppression action of Xuezhikang on immunity for its future use on GBS patients.Materials and Methods:1 AntigenThe antigen is the PO peptide 180-199.2 Induction and clinical evaluation of EANFemale Lewis rats, aged 6-8 weeks old, weighing 155-185g. The animal model was immunized by injection into both hind footpads with 200 ul emulsion containing 200 μg of P0180-199 peptide emulsified in an equal volume of complete Freund’s adjuvant containing 1.5 mg Mycobacterium tuberculosis. Before immunization (day 0 and thereafter everyday bodyweights and clinical scores were recorded and assessed as follows:0:normal,1:limp tails,2:mild hind limbs paralysis,3:moderate hind limbs paralysis,4:severe hind limbs paralysis,5:total tetraplegia.3 Xuezhikang treatment:Fifteen rats were randomly divided into three groups:control group, low-dose group and high-dose group. In each group there were five rats. In our study, Xuezhikang was administrated daily by gavage at the dose of 200 mg/kg/day and 1000 mg/kg/day from day 5 to day 16 post immunization, respectively. The control group was administrated with the same volume of 0.9%saline solution only in the same way4 Preparation of the lymph node mononuclear cells.The inguinal lymph nodes of rats were removed under aseptic conditions. Single-cell suspensions of mononuclear cells (MNC) from individual rat were prepared by grinding the organ with cell strainers in serum-free medium. Then, cells were washed three times and re-suspended to 2×106/mL in RPMI1640.5 Flow cytometric analysis of intracellular cytokines.For intracellular cytokines analysis, lymph node MNC were incubated with PE-labelled anti-rat-IL-10, PE-labelled anti-mouse/rat-TNF-a, FITC-labelled anti-mouse/rat IL-17A and FITC-labelled anti-rat-IFN-y for 30 min at 4℃ in the dark respectively. Then, cells were analysed using a FACScan.6 Flow cytometric analysis of Treg cells among MNC from lymph nodes.Fixation and permeabilization of lymph node MNC were performed using the eBioscience Foxp3 Staining Buffer Set. FITC-conjugated anti-rat CD4, PE-conjugated anti-rat CD25 and PE-Cy5-conjugated anti-mouse/rat Foxp3 antibodies were used for staining according to the protocol recommended by eBioscience.7 Statistical analysis.Statistical analysis of the data was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Differences between three groups were tested by one-factor analysis of variance (ANOVA) followed by least significant difference (LSD) test as a post hoc test. Results were expressed as mean standard deviation (SD), and a level of P<0.05 was considered significant.Results:1 Therapeutic treatment Xuezhikang decreased PO-induced EAN rats clinical scores.2 Xuezhikang suppressed the cytokines secretion of TNF-a, IL-10 and IL-17.3 Xuezhikang down-regulated the percentages of CD4+CD25+T cells and CD4+Foxp3+T cells among lymph node MNC.Conculsion:Xuezhikang could ameliorate EAN with suppression on immune system.