Neuroprotective Effect And Mechanism Of Hydroxyfasudil On MOG And CPZ-induced Demyelinating Mouse Model Of Central Nervous System

The Rho kinase(ROCK)signaling pathway is involved in numerous fundamental cellular functions such as cell migration,apoptosis,inflammation,adhesion and neurite outgrowth.ROCK was activated in the brain,spinal cord and spleen of EAE mice.A growing number of studies from our and other labs suggest that Fasudil ameliorated clinical symptoms of EAE,possibly by inhibiting inflammatory responses,shifting M1 microglia/macrophages toward M2 phenotype,promoting neuroprotection,synaptogenesis,axonal regeneration and the potential of endogenous neural stem cell mobilization.However,there are several limitations in the clinical practice of Fasudil,such as a relatively narrow safety window,lower bioavailability and a very short half-life.These limitations demonstrate that Fasudil is not suitable for long-term use in patients with MS or other chronic neurodegenerative diseases.Hydroxyfasudil(HF),active metabolite of Fasudil in vivo,exhibits higher biological activity and longer half-time period.HF is more selective than Fasudil as an inhibitor of ROCK.Animal experiments have shown that it has potential value in the prevention and treatment of malignant tumors,pulmonary hypertension,cardiovascular and cerebrovascular spasm,myocardial ischemia,ischemic brain injury and other diseases.Therefore,in the present study,we first observed the effect of HF on cell viability,toxicity in cultured SHSY-5Y neurons.Secondly,the safety of HF was verified by acute toxicity test.Then we tried to observe the therapeutic potential of HF in EAE.Finally,we compared the effect of HF and Fasudil in the Cuprizone(CPZ)induced demyelinating model,and explored the possible mechanisms.Part ? The effect of Hydroxyfasudil on cell viability,toxicity and morphology in SHSY-5Y neurons Objective:To observe and compare the cytotoxicity of HF and Fasudil,evaluate the effect of HF on the activity and morphology of SHSY-5Y neurons.Methods:The SHSY-5Y cell line was cultured in Dulbecco's modified Eagle's medium,supplemented with 10% fetal bovine serum,100 U/ml penicillin and 100?g/ml streptomycin at 37°C in a humidified cell incubator under a 95%/5%(v/v)mixture of air and CO2.Cells were plated on a culture dish(diameter=10mm)at a density of 1.0×105/ml.SHSY-5Y cells were respectively treated with Fasudil or HF at different concentrations(3?g,15?g,75?g and 200?g/ml)for 24 h.Cell morphology and spine formation were observed under a microscope.Cell viability of SHSY-5Y was measured by MTT assay.The levels of LDH release in supernatants of cultured cells were measured.Results:1.Compared with the PBS control group,relatively low concentrations(3?g/ml,15 ?g/ml)of HF and Fasudil were not cytotoxic.With increasing of drug concentration,especially when the concentration reached up to 200 ?g/ml,the toxicity to the cells was significant.Compared with the PBS group,the difference was statistical significance(P<0.05).There was no significant difference between HF and Fasudil.2.Compared with the PBS control group,relatively low concentrations(3?g/ml,15 ?g/ml)of HF and Fasudil did not affect cell viability.However,the cell viability were decreased when the concentration was increased up to 75 ?g/ml and 200 ?g/ml(P<0.05).There was no significant difference on cell viability and death between Fasudil and HF.These results show that both Fasudil and HF are safe at 3?g/ml and 15?g/ml in vitro,but cell activity decreased and cell death increased,with the increase of concentration.3.The synapses were observed after culturing with different concentrations of drugs for 24 h.Compared to the PBS group,the low concentrations of Fasudil or HF(3?g/ml)did not affect morphology of SHSY-5Y neurons.The concentration of 15?g/ml can promote the formation of protrusion effectively,but when the concentration is further increased upto 75?g/ml,the protrusions were damaged.Conclusion:HF was well tolerant in vitro and there was no significant difference compared with Fasudil.Part ? Acute toxicity study of Hydroxyfasudil in mice Objective:To observe the acute toxicity of HF in mice.Methods:Part 1:Thirty-six C57BL/6 mice(20–22g)were divided into 6 groups(3 male and 3 female per group)randomly,which were Fasudil-80,100,125mg/kg and HF-80,100,125mg/kg.Hydroxyfasudil and Fasudil were dissolved in DMSO before further dilution in 0.1% glacial acetic acid,and injected intraperitoneally.The final concentration of DMSO was 10%(v/v).Toxicity was observed in mice after the administration,including appearance,urine,feeding conditions,locomotor activity and death.Part 2:To test the influence of glacial acetic acid to the clinical appearances,the experiments of 100mg/kg and 125mg/kg were repeated.Sixteen mice were divided into 4 groups.Fasudil was dissolved in NS and HF was dissolved in 10% DMSO and diluted with 0.1% glacial acetic acid.Activities and toxic behaviors of mice were observed.Results:Part 1:With the increase of drug concentrations,spontaneous activities of mice gradually decreased,respiratory rate increased,the hind limbs can't stand.The convulsions occurred in mice treated with Fasudil-125mg/kg in 2 minutes after administration,followed by systemic rigidity and then died.The mice in the same concentration of HF group still showed more spontaneous activity without convulsions and deaths,and their respiratory rate did not increase significantly.Compared with HF group,mice in Fasudil group displayed less spontaneous activity,increased involuntary activity and higher incidence of convulsions and mortality at the same concentration.Part 2:Two of the mice in Fasudil-125mg/kg(dissolved in saline)group developed convulsions 5min after injection,the respiratory rate accelerated and died 30 min later.The remaining two showed less spontaneous activity with hind legs lying.In contrast,mice did not experience convulsions and deaths in the HF group-125mg/kg(dissolved in glacial acetic acid + DMSO),and three of them only showed less autonomic activity.Conclusion:The maximum tolerated dose of HF is greater than 125 mg/kg,indicating that HF is less toxic and safer than Fasudil.Part ? The protective effect of Hydroxyfasudil on MOG35-55 induced experimental autoimmune encephalomyelitis in C57BL/6 mice Objective:In this study,we try to observe the therapeutic potential of HF in EAE,and explored its possible cellular and molecular mechanisms.Methods:Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptides(MOG35-55),which were randomly divided into EAE group,HF group.on day 3 post immunization(p.i),HF was injected intraperitoneally at a dose of 40 mg/kg/day.The injection of saline was set up as control in a similar manner.Animals were weighted and evaluated for clinical score daily.On day 28 p.i,the frozen sections of the spinal cord were taken for histological examination,that is Hematoxylin-Eosin(HE)staining and Luxol Fast Blue(LFB)staining.Spinal proteins were extracted to evaluate the expression of NF-?B,COX-2 and i NOS by Western blot.Spleen mononuclear cell(MNCs)suspensions were prepared.In addition,we explored the direct role of HF in vitro.The splenocytes of MOG35–55-immunized mice were obtained on day 9 p.i.,and treated with HF(15 ?g/ml)in the presence of MOG35–55(10 ?g/ml)for 72 h.The influences of HF on inflammatory cytokine and chemokine were detected according to ELISA and Real-time PCR.The T cells subsets were analyzed by Flow cytometry.We also observed the effects of HF administration on macrophage transformation.Results:1.Behavioral changes: On the 12 th day after immunization,mice in the EAE group developed motor dysfunction successively.The average onset time was 16.38±4.98 days after immunization,and the clinical symptoms increased and the score gradually increased with time.100% of mice immunized with MOG35–55 peptide developed clinical symptom.The mean highest score of the clinical symptoms of the the EAE group was(3.84±1.09),and there was no measurable dyskinesia in the HF-treated group.The administration of HF prevented the clinical onset and dramatically inhibited the development of EAE compared with control EAE mice from day 16 to 28 p.i.The difference between the two groups was significant(P<0.01,P<0.001).2.Histological examination: Extensive CNS inflammatory infiltration was observed in spinal cords of control EAE based on HE staining.There were a large number of inflammatory cells around the small blood vessels in the white matter of the spinal cord of EAE mice,especially around the small veins,showing a "sleeve-like" change of blood vessels.Inflammatory cell infiltration area of EAE group accounted for(1.15±0.09)% of total white matter area,of,and infiltration of inflammatory cells in HF treatment group(0.64±0.18%)was significantly decreased.HF-treated mice had a significant inhibition in the extent of inflammatory infiltration in spinal cords compared with EAE control.The two groups were statistically significant(P<0.01).LFB staining revealed the loss of myelin in the white matter of the spinal cord of EAE mice.The loss of myelin in the EAE group accounted for(1.20±0.16)% of the total white matter area and(0.37±0.02)% in the HF-treated group,which significantly reduced the loss of myelin in the spinal cord(P<0.01).LFB staining showed that administration of HF significantly prevented demyelination associated with EAE in the spinal cord compared with control group.3.Flow cytometry was used to detect the effect of HF on the changes of T lymphocyte subsets in EAE spleen MNCs.The results showed that compared with the EAE group,HF could up-regulate CD4+CD25+ T cells(1.15±0.17 in the EAE group and 1.85±0.02 in the HF group,P<0.05),and down-regulate CD4+IFN-?+(9.93±1.65 in the EAE group,2.64±0.18 in the HF group,P<0.05)and CD4+IL-17+T(12.34±1.26 in the EAE group and 2.10±0.16 in the HF group,P<0.05).Levels of cytokine IFN-? and IL-17 were significantly suppressed after HF treatment(P<0.05).4.The treatment of HF obviously suppressed expression of i NOS CD68 and TNF-? compared to control EAE(P<0.001).Conversely,the treatment of HF effectively enhanced the expression of M2 marker Arg-1,IL-4 and TGF-? compared to control EAE(P<0.05 and P<0.01).5.HF significantly declined the expression of p-NF-?B /p65(P<0.05),and markedly inhibited the expression of COX-2 and i NOS(P<0.05 respectively)in the spinal cord compared to control EAE.6.The treatment of HF declined the production of IFN-? and IL-17(P<0.05 respectively),and decreased the release of inflammatory TNF-? and IL-1?(P<0.05).The treatment of HF decreased the levels of MIP-1a,MCP-1,RANTES m RNA as compared with control EAE(P<0.05 and P<0.01),Conclusion:HF delayed the clinical onset and suppressed the clinical severity of EAE,accompanied by the improvement of the associated pathological damage.This beneficial efficacy appears to be mediated by the following effects:(1)HF modifies immune out-of-balance induced by EAE;(2)HF shifts macrophage/microglia phenotype from M1 to M2,which should display the efficacy of anti-inflammation;and(3)HF inhibits the expression of chemotactic factors on the immune or inflammatory cells,reducing the migration of inflammatory cells into the CNS.Part ? The comparision of therapeutic effects on cuprizone-induced demyelination mouse model between Hydroxyfasudil and Fasudil and underlying mechanism Objective:To compare the therapeutic effect of HF and Fasudil on CPZ-induced demyelination mouse model and explore the possible mechanisms.Methods:Male C57BL/6 mice were divided into three groups,Normal group: received rodent chow;Cuprizone group: fed CPZ diet and injected with normal saline intraperitoneally(i.p.);Fasudil-treated group and HF-treated CPZ group,which received i.p.with Fasudil or HF(40 mg/kg/day)after 4 weeks and for consecutive 2 weeks,without CPZ withdrawal.Elevated plus-maze(EPM)test and Pole test were used to measure anxiety level and locomotor coordination of mice.To examine the extent of demyelination,slides of brain were stained with Luxol Fast Blue(LFB),Black gold ?(BG ?)staining.Protein from brains were extracted and MBP were detected by Western blot.Immunohistochemistry staining method was adapted in MBP and O4.Spleens were isolated and weighted.Suspensions of MNCs were incubated in the presence or absence of mouse MOG35-55(10?g/ml)for 48 h.Supernatants were harvested and measured for cytokine IFN-?,IL-10,IL-17,IL-6,TNF-? and IL-1?.The alternation of T cell responses,neuroinflammation and the neurotrophic factor by HF were observed.The concentration of anti-MOG antibody in serum,spleen MNCs culture supernatant and brain tissue was determined by ELISA and Dot blot.Brain sections were used to perform immunohistochemical staining with primary antibodies: anti-CD4,anti-CD68,anti-CD220,anti-Iba-1,anti-i NOS,anti-NF-?B,anti-BDNF.T cell subsets of splenic MNCs were detected by flow cytometry.Results:1.Compared with the normal group,the CPZ model group showed obvious behavioral changes.The EPM test showed that the number of mice entering the closed arm in the CPZ group increased significantly(normal group was 4.75±0.93,CPZ group was 10.08±1.89,P<0.01).Compared with the CPZ group,the Fasudil group(6.58±1.05,P<0.05)and the HF group(5.83±0.79,P<0.05)effectively reduced the number of times the CPZ mice entered the closed arm.There were no significant differences between the Fasudil group and the HF group.The results of Pole test showed that the time from the top to the bottom of the detection device in the CPZ group was significantly higher than that in the normal group,which was(5.72±0.83)s in the Normal group and(15.29±1.24)s in the CPZ group,P<0.01.Compared with the CPZ group,the Fasudil group was(11.71±1.01)s,P<0.05,and the HF group was(10.14±1.10)s,P<0.05,which effectively improved the exercise capacity of CPZ mice.However,the difference between the Fasudil group and the HF group did not reach statistical significance.2.The intensity of LFB staining,BG II staining and MBP was obviously decreased in the corpus callosum(CC)region of CPZ-fed mice,as compared to normal mice without CPZ feeding(P<0.001).Fasudil or HF treatment significantly attenuated CPZ-induced demyelination in the CC region(P<0.01,P<0.001).The intensity of BG II in Fasudil group was higher than HF group(P<0.05).3.In Fasudil and HF group,an increased expression of MBP protein was observed in brain by western blot(compared with CPZ group,P<0.001).The immunofluorescence staining of O4 antibody also indicated that O4+ mature oligodendrocytes were higher in Fasudil and HF group than that in CPZ group(P<0.001).4.The spleen quality of the Normal group and the CPZ group were(113.50±14.92)g and(57.13±2.65)g.The CPZ group was significantly lower than that of the Normal group(P<0.001).The spleen mass of the Fasudil group was(70.77±5.16)g,and that of HF group was(65.83±3.04)g.Compared with the CPZ group,both of them could partially improve the spleen atrophy induced by CPZ(P<0.05).There was no significant difference between the Fasudil group and the HF group.5.In addition,Fasudil and HF treatment effectively decreased the titer of MOG antibody in serum,supernatants of cultured splenocytes and extracts of brain by ELISA and dot blot.The effect of inhibition on MOG antibody was stronger in HF group than in the Fasudil group(P<0.05).The expression of anti-MOG antibody in serum of different dilution ratios was tested by Dot blot.The anti-MOG antibody in CPZ group was significantly higher than normal group(P<0.001),while Fsudil and HF inhibited the level of antibody(P<0.001,P<0.01),HF was more effectively than Fasudil(P<0.05).The specificity of the anti-MOG antibody was also examined by Dot blot.The MOG antibodies penetrated into the brain can bind to oligodendrocytes and lead to the damage of oligodendrocytes.6.Flow cytometry was used to analyze the proportion of B lymphocyte subsets in spleen MNCs.The proportion of B220+B cells in Normal group,CPZ group,Fasudil group and HF group were:(52.37±0.52)%,(62.17±0.13)%,(54.87±0.59)%,(52.97±0.23)%,the CPZ model group was increased compared with the Normal group(P<0.001).Compared with the CPZ group,Fasudil and HF inhibited this change(P< 0.001).The HF group was lower than that of Fasudil group,and the difference was statistically significant(P< 0.05).In the brain of CPZ model mice,the expression of CD4,CD68 and B220-positive T lymphocytes,macrophages and B lymphocytes were observed,and the difference was significant compared with the Normal group(P<0.001).Fasudil and HF significantly inhibited the infiltration of these cells into the brain of CPZ mice(P< 0.001).7.Compared with the Normal group,the number of Iba-1+ microglia/macrophages in the CC region and striatum of CPZ mice was significantly increased(P<0.001).Compared with Fasudil and HF group,the expression of this cell in the brain of CPZ mice was effectively reduced(P<0.001).There was no significant difference between the Fasudil group and the HF group.Using double staining technique,the number of Iba-1+i NOS+ and Iba-1+NF-?B+ in the brain of CPZ group were significantly higher than that in the Normal group(P<0.001).Fasudil and HF effectively reduced the number of inflammatory cells in the brain of CPZ mice,and the difference was significant compared with the CPZ group,P<0.001,while there was no significant difference between Fasudil and HF groups.Fasudil and HF inhibited the levels of inflammatory cytokines IL-4(P<0.001),IL-1?(P<0.001)and TNF-? in the brain of CPZ mice.8.HF administration induced an increased BDNF immunoreactivity that co-localized with GFAP+ astrocytes.Conclusion:HF and Fasudil inhibited microglia-mediated neuroinflammation by inhibiting the activation of microglia and M1-polarization in the CPZ-induced demyelination.In addition,HF and Fasudil inhibited the MOG antibody and prevented the infiltration of inflammatory cells.HF treatment promoted the production of astrocyte-derived BDNF,which should contribute to the protection and regeneration of myelin sheath.Furthermore,HF showed a stronger inhibitory effect on anti-MOG antibodies than Fasudil.Conclusion1.The cytotoxicity of HF and its effects on cell activity and cell morphology are concentration-dependent.2.The maximum tolerated dose of HF is higher than Fasudil,which has better safety.3.HF can improve the clinical symptoms of EAE model,reduce the infiltration of inflammatory cells in the spinal cord,improve the loss of myelin,and play a role by regulating immunity and inhibiting neuroinflammatory reactions.4.HF and Fasudil can protect the CPZ model and promote remyelination by inhibiting the production of MOG-specific antibodies,inhibiting microglia-mediated neuroinflammation,improving the inflammatory microenvironment,and promoting the production of neurotrophic factors.5.HF and Fasudil showed similar effects in the treatment of CPZ model,but compared with Fasudil,HF has stronger inhibition to MOG-specific antibodies.With the advantages of lower toxicity and lower adverse reactions,HF has great potential application value.