The Mechanism Study And Treatment Effect Of Yinxieling On Multiple Sclerosis

ObjectiveMultiple Sclerosis(MS)is a chronic and progressive disease characteristic by demyelination changes in central nervous system(CNS)white matter.Predilections of this disease are young adults,more common in women,which is the most common cause of non-traumatic neurological disability.Because of the high rate of recurrence,chronic course for the society heavy medical,psychological and economic burden and seriously affecting the quality of life,the disease has been highly valued.The clinical characteristics of the lesions show widely disseminated and simultaneously or sequentially involve the brain,cerebellum,brainstem and spinal cord.The course of the disease is often intermittent.The brain,spinal cord and optic nerve are progressive damaged.MS in pathology are mainly as four aspects:inflammation,demyelination,axonal damage and glial cell proliferation.The cumulative distribution of inflammatory cells in the spinal cord white matter and perivascular,oligodendrocytes,neurons and axonal damage are formed extensive demyelination manifestations in white and gray matter area of cerebral and cerebellar cortex,thus affecting the entire central nervous system and cause transient neurological damage or permanent loss,severe cases may eventually disability.It is generally believed that MS occurs on the basis of susceptibility genes,immune system itself has been adversely influenced by environmental factors and certain viral infections,resulting in nerve inflammationn and marrow sheath impaired.Chronic inflammation and broken of auto-reactive T cells in immune tolerance are the main features of MS in immunology,especially the Th cell subsets imbalance in CNS.Therefore,from the perspective of regulatory T cell subsets balance,especially the Th cell subsets,it is important to in-depth understand multi-target pathogenesis for the further elucidate the immune mechanism of MS.Yinxieling tablets is a prescription of famous Chinese medicine and dermatologist Professor Xuan Guowei with his clinical application experience for decades,which was issued formulation approval by the Guangdong Provincial Food and Drug Administration in 1998(Z20080123)(Patent publication number:CN 105233150A).Chloranthus spicatus,curcuma zedoary,smoked plum,rhizoma smilacis glabrae,radix paeoniae rubra,lithospermi and liquorice constituted the main ingredient of this tablet and gathered the clinical efficacy on exact treatment of psoriasis.The optimized formulation is the result for the original prescription compatibility seeking out the best treatment of psoriasis vulgaris.The composition of chloranthus spicatus,curcuma zedoary,smoked plum,rhizoma smilacis glabrae,radix paeoniae rubra,lithospermi and liquorice was patent protected in 2009,publication number was CN 101632827A.This recipe abided by the law of nourishing and moistening,cooling blood detoxification and circulation collateral,brought the exact effect especially to blood stasis,blood deficiency and wind-dryness psoriasis.Various studies show that Yinxieling therapeutic mechanism of autoimmune diseases may be regulating T cell subsets imbalance,suppressing abnormal humoral immunity to balance the body's humoral and cellular immunity,improving microcirculation lesions and inhibiting inflammation.Therefore,this paper intends to regulate T cell subsets from the viewpoint of the balance to observe the Yinxieling tablets and optimized formulation therapeutic effect of MS,and explore its mechanism of action,not only contribute to a better understanding of the immune mechanisms of MS,but also expanded the therapeutic range,which provide evidence for scientific explanation of Yinxieling pharmacodynamic material basis.Methods1.Therapeutic effect of Yinxieling tablets and optimized formulation on EAE-induced by myelin oligodendrocyte glycoprote(MOG35-55).(1)Forty female C57BL/6 mice were randomly divided into three groups,normal control group(Control),EAE model group(Model),Yinxieling tablets treatment group(YXL-10P)and Yinxieling optimized formulation treatment group(YXL-7S).In addition to the Control group,the mice in other two groups were injected by subcutaneous MOG35-55 emulsifier to induce EAE model(Injection ratio:MOG35-55 200?g,M.T./CFA 500?g,P.T 300ng each mouse).(2)All mice were administered prophylactically from the day before immunization,once a day,YXL-10P group were given suspension orally(1.5g/kg),while YXL-7S group were gavaged with 15g/kg concentration.Model and control group were received doses of distilled water,continuous intragastric administration for 21 days.The dose is obtained in terms of the amount of the human body.Each group of mice was observed by mental status,body weight change and the rates of incidence everyday,while evaluated for neural function by Benson score on a scale from 0 to 5.(3)The brain white matter and enlarged lumbar spinal cord were harvested on the 21th day after post-immunization:H&E staining drawn with 10%chloral hydrate anesthetized by intraperitoneal injection in mice,cut the chest,complete removal of tissue fixed in 10%neutral formalin solution for 24 hours,paraffin-embedded sections to prepare,3.5 ?m sections for H&E staining to Okuda histopathological score,and luxol fast blue staining to analysis severity of demyelinating changes.Another part of fresh tissue were coverage in frozen embedded solution(OCT)quickly put cooling in liquid nitrogen,-80? stored for use,5 u m frozen sections for immunohistochemistry to quantified expression of CD3.(4)Part of the tissues were treated to be tested following requires of real-time quantitative PCR method to detect expression levels of inflammatory cytokines,such as IL-17A,IFN-y,ROR-yt,IL-6,TGF-?,IL-4,and IL-10 mRNA in brain and spinal cord tissue of mice.2.Inhibitory effect of Yinxieling tablets and optimized formulation containing serum on CD3 + T cell proliferation induced by concanavalin A(ConA).(1)The purified CD3+ T cells were respectively treated with Yinxieling tablets and optimized formulation-containing serum in complete RPMI 1640 medium of multiple concentrations(0,1%,5%,10%,20%).After 48h or 72h,MTS cell proliferation assay kit was used to test relative survival of CD3+ T lymphocytes and analysis security concentration of two Yinxieling containing serum(0-20%).(2)T cell proliferation model was established with a final concentration of 2?g/mL of ConA stimulation.Then different concentrations of serum containing Yinxieling tablets and optimized formulation was separately added into each well(concentration of 1%,5%,10%and 20%)for intervention 48 and 72h.MTS cell proliferation assay kit was used to test relative survival of CD3+ T lymphocytes,analyzed effective concentrations and onset time of two Yinxieling containing serum(1-20%).(3)CFSE-labeled cells were washed in PBS with 5%FBS/PBS twice after collection,and resuspended in PBS buffer.The percentage of total CD3+ T cells and divided CD3+ T cells were determined from the CFSE profiles on a FC500 flow cytometer,using CXP software(Beckman).(4)Fluorescence quantitative polymerase chain reaction(quantitative Realtime Polymerase Chain Reaction,quantitative rt-PCR)was applied to detect expression levels of inflammatory cytokines,such as IL-17A,IFN-?,ROR-?t,IL-6,TGF-?,IL-4,and IL-10 mRNA in cells.Results1.Therapeutic effect of Yinxieling tablets and optimized formulation on EAE models induced by MOG35-55(myelin oligodendrocyte glycoprotein)(1)Throughout the experiment,there is no incidence in control group,the reaction,activity and appetite of mice were normal,weight gradually increased.Seven days after immunization,induced mice gradually emerged clinical symptoms of listlessness,lags in response,decreased activity and significant neurological signs:tail drooping weakness,paralysis of hind limbs,some seriously,limbs are paralyzed and even showed agonal state.The manifestations are in line with the performances of EAE mouse model,and gradually get worse with time.The peak of outburst occured in the Day 17th,then neurological damage began to recover.Three groups of mice after MOG antigen,the incidence rate can reach 100%(P<0.0001),and no significant difference between Yinxieling tablets and optimized for:mulation with model group(10P:P=0.5454>0.05;7S:P=0.3710>0.05),which demonstrated successful modeling.Observed weight of mice,from 7 days after the induction of antigen to the Day 14th,obvious weight reduced of mice was not found in EAE model group compared with Con and YXL-7S group(P=0.0528>0.05).No significant difference suggested that YXL-7S play no effect on weight in the initial phase of onset.But there was lighter weight of mice in Yinxieling tablets administered group(YXL-10P)than the model group(P=0.0018<0.01),the presence of significant difference,the possibility reason still existed that the liquid was not completely dissolved particles,causing the mice gastrointestinal dysfunction that was satisfied by the decomposition,eventually leading to loss of appetite,than weight loss.However,the peak incidence in Day 17th,all the mice progressed to varying degrees,MOG-induced model group were significantly lower weight(P<0.0001),and after YXL tablets and optirmized formulation intervention,no obvious weight reduced,even heavier than Con(10P:P=0.003<0.01;7S:P<0.0001).Significant difference by statistical analysis was remindered that both YXL tablets and optimized formulation enable to make weight gain in late period of acute onset.However,pre-treatment with Yinxieling tablets and optimized formulation aqueous solution,disease severity in mice was lighter than the model group,the neurological score decreased obviously(10P:P=0.0482<0.05;7S:P=0.0244<0.05),which was statistically significant and indicated two Yinxieling decoction may evidently improve nerve damage functions of EAE mice.Some abnormal changes were found in the brain and spinal cord tissue,chiefly different degrees of perivascular inflammatory cell infiltration,small blood vessels dilated,partly surrounded by inflammatory cells surrounding capillaries,forming a typical"vascular cuff" change(P<0.01).Pathological features,such as glial cells demyelinating(10P and 7S group P<0.05),karyopyknosis,denaturation and neurons vacuolar necrosis,changed distinctly in the white matter region,lateral hippocampus and spinal cord white matter,part of the junctional zone between cortex and medulla may also occur.Moreover neurological score were high,the scope of inflammatory lesions and extent of cell infiltration were corresponding increased.While compared with those in the model group,there were also visible inflammatory cell infiltration and a few neurons vacuolar necrosis in the brain and spinal cord tissue,but the extent of histopathologic changes were alleviated to various degrees,e.g.,a smaller number of infiltrative cells,better recovery of neuron injury and no significant "vascular cuff "change.Results showed that CD3 protein expression both in the model and Yinxieling tablets or optimized formulation group was significantly higher than that in the control group on day 21.Furthermore,IOD of CD3 expression in the model group was significantly higher than that in YXL tablets and optimized formulation group(P<0.05 in brain and P<0.01 in spinal cord).(2)The gene expression of pro-inflammatory cytokines,like CD3?,IL-17A,ROR-?,IFN-y and IL-6 in white matter and spinal cord markedly up-regulated in mice of model group compared with that of control group on the 21th day immunization.Compared with that of model group,IL-17A,ROR-y,IFN-y and IL-6 mRNA expression decreased in YXL tablets and optimized formulation groups(P<0.05),especially for IL-17A mRNA in spinal cord of mice in YXL-10P group(P=0.0043<0.01).There was a statistical significance.The anti-inflammatory factor expression of TGF-?31 IL-10 and IL-4 mRNA in white matter and spinal cord obviously decreased in the model group compared with the control group on the 21th day after induced EAE(P<0.05),while the level of g,ene expression significantly increased in spinal cord by the treatment of YXL tablets and optimized formulation compared with the model group(P<0.01).Descriptions suggested Yinxieling tablets and its optimization can reduce the Th cells,especially Thl and Th17 cell infiltration in the lesion tissues.2.Inhibitory effect of Yinxieling tablets and optimized formulation containing serum on CD3+ T cell proliferation induced by concanavalin A(ConA).(1)Viability of CD3+ T cells in spleen were not affected after a 48h-or 72h-incubation with 0 to 20%Yinxieling tablets and optimized formulation containing serum compared with the blank rat serum,indicating that two Yinxieling-containing serum had no toxicity on CD3+ T cells.(2)After ConA stimulation,CD3+ T cells proliferated obviously as indicated by the increased percentages of total and divided T cell.By contrast,YXL tablets and its optimized formulation serum evidently decreased the percentage of the total T cells,at the same time reduced the proliferation proportion of total T cells.Furthermore,CD3+ T cells proliferation was inhibited by different concentrations of containing serum in varying degree,even 20%YXL tablets and optimized formulation significantly deceased to more than 50%(39.37%in 10P and 43.15%in 7S group).It was prompted that 20%Yinxieling containing serum significantly inhibited proliferation of CD3+ T cells in mouse spleen stimulated by conA(10P:P<0.0001;7S:P = 0.0029<0.01).(3)Through CFSE fluorescence labeled,we found T cell proliferation and strongly split were stimulated by ConA,on the contrary,YXL tablets and optimized formulation-containing serum could inhibit the division,in which 20%YXL optimization serum reduced chi-square attenuated 70%(P<0.0001).By Annexin V-PI staining,it was found that no apoptosis was observed in the ConA-stimulated CD3+ T cells treated by 5%,10%and 20%Yinxieling tablets and its optimized formulation-containing serum.All these suggested that two YXL-containing serum could inhibit ConA-stimulated T cell proliferation directly,but not via the way of apoptosis.(4)In addition,the cytokine profile indicated that,levels of the inflammation cytokine(pro-inflammatory IL-17A,IL-6,IFN-y and anti-inflammatory IL-4)soared to varying degrees after ConA stimulation for 72h.Yinxieling tablets and optimized formulation-containing serum had impacts statistically on these cytokines(P<0.05).Upon the stimulation,Yinxieling optimization-containing serum inhibited the releases of anti-inflammatory IL-10,TGF-? obviously(P<0.01).The two Yinxieling serum had less impact on the transcription factor ROR-?t,but there was still a downward trend(P<0.05).ConclusionsTaken together,Yinxieling tablets and optimized formulation can both significantly improve the quality of life of EAE mice,relieve the inflammation in central nervous system of sick mice,reduce blood vessels to dilate lesions,extenuate demyelination and neuronal necrosis,decrease infiltration of inflammatory cells,reduce " vascular sets of "formation,these indicate Yinxieling has a therapeutic effect on EAE.In vitro results further suggest that a therapeutic effect of Yinxieling may be directly related to immunosuppression.Yinxieling tablets and optimization containing serum can directly inhibit proliferation of T lymphocytes,reduce the secretion of pro-inflammatory cytokines and promoting anti-inflammatory cytokines,regulate balance of Th lymphatic cell subsets and finally passivate adaptive immune response to weaken the development process of aggressive immune response in EAE/MS.The results above suggest that Yinxieling tablets or optimized formulation has a significant therapeutic effect on EAE,which is an animal model of multiple sclerosis.The role is closely related to its direct inhibition of T cell proliferation and detente of adaptation inflammatory response in EAE acute phase.Those reveal potential application of Yinxieling presence in the treatment of multiple sclerosis.