Multiple sclerosis (MS) is one of the most common demyelinating diseases of the central nervous system, with pathological hallmarks including the destruction of myelin, axonal damage, and oligodendrocytes death.1 Among young adults, it is the most common non-traumatic cause of neurological disability. The pathogenesis of MS is complex and existing MS therapies are partly effective, thus combination therapy in MS is an attractive treatment strategy.2 Experimental autoimmune encephalomyelitis (EAE) is the principal model of MS.3 Lots of novel treatments about MS were experimented in EAE.
Ulinastatin (UTI) is a multivalent Kunitztype serine protease inhibitor found in human urine and blood. With anti-inflammatory and antiprotease activity, UTI has been widely used in inflammatory diseases such as severe sepsis, acute pancreatitis and so on.4–6 And UTI also had been documented to protect against cerebral ischemia-reperfusion injury.7,8 Recently, we reported that UTI has protective effects against EAE, which partly attributed to the protection against oligodendrocytes (OLGs) apoptosis and demyelination.9
Methylprednisolone, as other glucocorticoids, exerts powerful anti-inflammatory and immunosuppressive activities and has been the standard therapy of acute MS relapse.10 Then combination strategies of methylprednisolone and other drugs have been proved to be desirable.11–14 At present, no experiment was reported in literature, in which methylprednisolone combined UTI was used in the treatment of MS/EAE. In this paper, we investigated whether or not combination treatment with UTI and methylprednisolone exerted protective effect in EAE mice.
EAE induction and clinical evaluation
Six to eight-week-old female C57BL/6 mice weighing 16–18 g were purchased from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of Sun Yat-sen University. EAE induction was performed referring to previously published protocols.9 In brief, on day 0 mice were immunized subcutaneously in the flanks with 200 μg of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (CLBIO-SCIENTIFIC Co., Ltd, Xi'an, China; purity >95%) (per animal) was emulsified in complete Freund's adjuvant (CFA, Sigma-Aldrich, USA) containing 500 μg Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI). Furthermore, 400 ng pertussis toxin (PTX, Alexis Corp., San Diego, CA, USA) in 100 μl PBS was intraperitoneally injected on days 0 and 2, and MOG35–55 peptide in CFA was administered again on day 7. Clinical scores were defined as follows: 0 = no sign; 1 = loss of tail tonicity; 2 = flaccid tail; 3 = ataxia and/ or paresis of the hind limbs; 4 = complete paralysis of the hind limbs; and 5 = moribund or death.
Animal groups and treatment
Mice were randomly divided into UTI treatment group (group U), normal saline treatment group (group S), normal control group (group N, the mice were not immuned), methylprednisolone treatment group (group P) and combined treatment with UTI plus methylprednisolone (group U+P), with n=8 in each group. Treatment was started on day 12 post immunization. UTI (Techpool Biochem, Guangdong, China) was intraperitoneally injected into each mouse in group U, once daily for 10 days, at a dose of 250 000 U/kg after EAE onset. In group P, methylprednisolone (Pfizer manufacturing Belgium NV) was given at a dose of 30 mg/kg, i.p, once daily for 5 days. UTI (250 000 U/kg per mouse, once daily for 10 days) and 30 mg/kg methylprednisolone (30 mg/kg per mouse, once daily for 5 days) were intraperitoneally administered in group (U+P) mice. Mice in groups S and N were intraperitoneally administered 0.1-ml saline per mouse at the same time and for the same course as group U. Mice in each group were sacrificed at day 35. Eight mice in each group were included and three similar experiments were performed.
At the time of sacrifice (at day 35), mice were anesthetized with 10% chloral hydrate. Histopathological technique was described previously.9 In brief, the mice anesthetized were perfused via cardiac puncture from the left ventricle with ice cold PBS, and followed with 4% paraformaldehyde. Then the lumbar spinal cords were removed, postfixation in the same fixative, embedded in paraffin, sectioned (4 μm thick), and stained with solochrome cyanin impregnation to reveal demyelination. Demyelination in the spinal cords was scored as previously described.15 1 = traces of subpial demyelination; 2 = marked subpial and perivascular demyelination; 3 = confluent perivascular or subpial demyelination; and 4 = massive perivascular and subpial demyelination involving one half of the spinal cord with the presence of cellular infiltration in the central nervous system (CNS) parenchyma; 5 = extensive perivascular and subpial demyelination involving the whole cord section with the presence of cellular infiltrates in the CNS parenchyma.
Western blotting analysis
The brains were harvested from the anesthetized mice and the cerebral cortices were isolated quickly and stored in -80°C until needed. Western blotting technique was described previously.9 In brief, proteins were extracted with RIPA lysis buffer (Keygen, Nanjing, China). Equal amounts of denatured proteins determined by the BCA protocol (Keygen) were loaded and analyzed by SDSpolyacrylamide gel electrophoresis and immunoblotting following standard protocol. The denatured proteins were transferred to PVDF membranes (Millipore, Billerica, USA) and blocked for 2 hours with 5% milk (non-Fat powder, MBCHEM, Shanghai, China) in TBS (Tris-bufferedsaline, Boster Biotechnology, Wuhan, China) containing 0.1% Tween (AMRESCO). Then the membranes were incubated overnight at 4°C with primary Abs that were diluted in 5% BSA (MBCHEM). The primary antibodies were anti-CNPase (Chemicon, USA, MAB326, 1:1000), anti-myelin basic protein (MBP) (Enzo-Biomol, UK, SA-601, 1:500), anti-proNGF (Epitomics, USA, EP1318Y, 1:1000), anti-p75 (Chemicon, USA, AB1554, 1:1000), and anti-iNOS (Millipore, USA, AB5328, 1:1000). Anti-β-actin (Bios, bse-0295G, Beiking, China, 1:1600) was chosen as a standard. After three washes (10 minutes each time) with TBST, the membranes were incubated for 1 hour with the appropriate horseradish peroxidase (HRP)-conjugated secondary Abs. Then washed again and developed with an ECL kit (Chemiluminescent HRP substrate, Millipore, WBKLS0100). The bands were scanned by GS-800 Calibrated Densitometer (Bio-Rad, Berkeley, USA) and analyzed by Quantity-one software (Bio-Rad).
Data were expressed as mean ± standard error (SE). Values were analyzed by SPSS 16.0 software (SPSS Inc., Chicago, USA). Differences between clinical scores and histological scores were analyzed with Mann-Whitney tests. Differences between protein levels (CNP, MBP, proNGF, p75, and iNOS) were analyzed with one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc tests. A P <0.05 was considered statistically significant.
Combined treatment alleviated EAE and reduced clinical scores
The clinical scores of combined treatment group with UTI combined methylprednisolone (group U+P, 0.61±0.06), UTI treatment group (group U, 0.97±0.06), and methylprednisolone treatment group (group P, 0.87±0.06), respectively, were significantly lower than the group with normal saline treatment (group S, 1.39±0.08, P <0.001). And the combined group had significantly lower clinical scores than groups with UTI or methylprednisolone treatment (Figure 1A and 1B).
Combined treatment reduced demyelination
Demyelination in the normal saline treatment group (group S) was the worst compared to the other groups (Figure 2A). The scores of demyelination in combined treatment (group (U+P), 1.33±0.33) were lower than that in methylprednisolone treatment (group P, 1.67±0.33) and saline treatment group (group S, 2.75±0.49), and there is a statistical significance between groups (U+P) and S (P=0.024). And there were no significant differences of the clinical scores between groups with UTI combined methylprednisolone and UTI or methylprednisolone treatment (Figure 2B).
Combined treatment enhanced expressions of CNP in the brain
Expression of CNP protein in combined treatment group was, respectively, higher than methylprednisolone treated group (group P) and saline treated group (group S), and there was a significant difference between groups (U+P) and S (P <0.001), but difference between groups (U+P) and P was not significant (P=0.379). In addition, CNP expressions in groups treated with UTI combined methylprednisolone, UTI, and methylprednisolone were, respectively, significantly lower than that in normal control group (group N, Figure 3A and 3B).
Combined treatment enhanced expressions of MBP in the brain
MBP protein expression in combined treatment group (group U+P) was, respectively, higher than groups treated with saline (group S), methylprednisolone (group P), and UTI (group U), and the difference between combined treatment group and groups P or S was significant (Figure 4A). Besides, the protein expressions in groups U, P, and normal control group (group N) were all significantly higher than that in group S (Figure 4A and 4B).
Combined treatment reduced expressions of proNGF protein in the brain
Compared to saline treated group (group S), the expressions of proNGF in the brains of combined treatment group, UTI treated group (group U), methylprednisolone treated group (group P) and normal control group (group N) were all significantly decreased. And expression of proNGF in combined treatment group was, respectively, significantly lower than that in groups U and P (P <0.05). Besides, there was no significance between groups P and U, groups U+P and N (Figure 5A and 5B).
Combined treatment reduced expressions of p75 protein in the brain
Expression of p75 protein in combined treatment group (group U+P) was lower than saline treated EAE group (group S), methylprednisolone treated group (group P) and UTI treated group (group U), however, only difference between group (U+P) and group S was significant. And p75 expressions in groups P, U, and N were significantly lower than that in group S. There was no significant difference between groups P and U (Figure 6A and 6B).
Combined treatment reduced expressions of iNOS protein in the brain
Expression of iNOS protein in combined treatment group (group U+P) was, respectively, lower than groups U, P, and S, and the difference between groups (U+P) and P or S was significant. iNOS protein expressions in groups U and P were all significantly lower than group S, but the difference between groups U and P was not significant (Figure 7A and 7B).
More and more literatures11,14,16,17 supported that combined therapy in MS or EAE achieved a better therapeutic response. As we know, methylprednisolone has been widely used for MS/EAE with its powerful anti-inflammatory and immunosuppressive activities. Though few papers reported that UTI was useful in treatment of MS/EAE, UTI was shown to be responsible for multiple effects in other diseases animal models, such as exerting neuroprotective effect in a rat model of cerebral ischemia/reperfusion injury,8 alleviating neuroinflammation in aged rats following partial hepatectomy,18 suppressing inflammatory response and lymphocyte apoptosis in septic mice5 and so on. And we recently have demonstrated that UTI has neuroprotective effects in EAE mice.9 Under the hypothesis that protective influences might be complementary to each other if both substances were combined, we tested the combined effect of methylprednisolone and UTI in EAE in this study. In the present paper, we found that UTI combined methylprednisolone evidently alleviated EAE clinical severity compared to monotherapy.
Oligodendrocytes (OLGs) generated myelin sheaths. Damage to OLGs could result in demyelination and neurological disability. 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) was an important marker protein of OLGs,19 myelin basic protein (MBP) was chosen as a marker of myelin sheath.20 In our previous study, we found that a large amount of OLGs death and demyelination occurred and markedly CNP, MBP expression decreased in the cerebral cortices of EAE mice, while UTI could inhibit OLGs death and demyelination, and increase CNP, MBP expression.9 And some papers reported that methylprednisolone could protect oligodendrocytes21,22 and promote remyelination.23 Based on the above basis, we explored the synergistic effect of UTI combined methylprednisolone in EAE for further study. Interestingly, we found that UTI combined methylprednisolone could markedly increase expression of MBP in cerebral cortices compared to monotherapy in the present paper. However, expression of CNP in UTI combined methylprednisolone treatment EAE mice was only significantly higher than that of saline treated EAE mice. Therefore, our results suggested UTI combined methylprednisolone could increase the expressions of MBP, CNP in cerebral cortices in EAE, furthermore, UTI and methylprednisolone exert synergic effects in increasing MBP protein expression.
OLGs and neurons apoptosis played an important role in MS/EAE pathogenesis. Some papers showed that UTI could decrease intestinal mucosal cells apoptosis24 and suppress lymphocyte apoptosis.25 however, few literatures reported the effect of UTI on OLGs and neurons apoptosis in the central nervous system. The precursor form of NGF (proNGF), binding to p75NTR, facilitated a cell death signaling cascade and promoted OLGs and neurons apoptosis.26,27 UTI could down-regulate the proNGF and p75 proteins expression in EAE in our previous study.9 In this paper, proNGF in UTI combined methylprednisolone treatment EAE was significant lower than that of monotherapy (UTI or methylprednisolone), however, p75 protein expression was only significantly lower than that of saline treatment EAE. As a result, combination treatment with methylprednisolone and UTI reduced proNGF, p75 in EAE, what's more, methylprednisolone and UTI synergistically reduced proNGF expression in EAE.
Nitric oxide (NO) has been implicated in the etiopathology of MS and EAE.28–31 Inducible nitric oxide synthase (iNOS) was one of the three nitric oxide synthase (NOS) isoforms that catalyzes the formation of NO from L-arginine. iNOS was induced in response to inflammatory stimuli. Increased expression of iNOS mRNA was found in EAE,32 and inhibition of iNOS could ameliorate the induction of EAE.33 At present, lots of studies had demonstrated that UTI could not only suppress systematic inflammation5,34–37 and but also alleviate neuroinflammation.18 Recently, Hua et al38 reported that UTI could effectively inhibit the increased expression of iNOS in degenerated nucleus pulposus cells of rabbits. Sung et al39 demonstrated that UTI suppressed nitric oxide production via the suppression of iNOS expression through the down-regulation of NF-kappaB activity in BV2 mouse microglial cells. These data suggested that UTI could exert anti-inflammatory effects and inhibit iNOS expression. In the present paper, expression of iNOS protein was increased in EAE brains and UTI, methylprednisolone, and UTI combined methylprednisolone all could downregulate iNOS expression in EAE mice brains. Meanwhile, UTI combined methylprednisolone had synergic effect in reducing iNOS expression.
In our previous findings, the dose of 250 000 U/kg UTI treated EAE showed protective effects through protection against oligodendrocyte apoptosis and demyelination through up-regulation of NGF and BDNF expressions and down-regulation of proNGF and p75.9 In the present study, the same dose of UTI (250 000 U/kg) was adapted to EAE groups with UTI, UTI combined methylprednisolone treatment. And UTI also showed the ability to protect against EAE through increasing CNP/MBP protein expressions and decreasing proNGF/p75/iNOS expression. And we mainly focused on the synergic effects of combination with UTI and methylprednisolone in EAE.
We acknowledge that there were several limitations in our experiment. First, UTI combined methylprednisolone protect against EAE possibly through multiple pathways such as anti-inflammation, immunoregulation, cytoprotection and so on, but we only investigated the expressions of CNP\MBP\proNGF\p75\iNOS proteins in EAE brains. Second, to evaluate the demyelination in EAE, we only used Solochrome cyanin staining, and in future studies we should add the Luxol Fast Blue and Bielshowsky (LFB+B) staining to show the myelin and axons. Third, as a result of NO/iNOS pathway, which plays an important role in pathological mechanism of MS/EAE, we should detect the NO level in EAE mice brain in future study.
In conclusion, UTI combined treatment with methylprednisolone could attenuate EAE clinical severity, decrease demyelination and protect against EAE. This property may be partly through up-regulating MBP, CNP expressions and down-regulating proNGF, p75 iNOS expressions. Furthermore, UTI combined methylprednisolone could exert synergic effects in increasing MBP expression and decreasing proNGF, iNOS expressions. Our results suggest that the combination therapy might be a novel potential treatment in MS.
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