Study Of Therapeutic Effect Of ZSTK474 On Experimental Autoimmune Neuritis

Background and Objective:GBS?Guillain-Barrésyndrome?is an autoimmune neuropathy that causes myelination and inflammatory cell infiltration of the peripheral nervous system?PNS?,which can be expressed as limb symmetry flaccid paralysis.Involving respiratory muscle patients can be fatal,and some patients left serious neurological deficits.Experimental autoimmune neuritis?EAN?is a classical animal model of GBS.The helper T cells?Th1/Th17?cells are essential participants in the inflammatory and immune processes of experimental autoimmune neuritis?EAN?.SinceTh1/Th17 cells are pro-inflammatory cells and both secrete pro-inflammatory cytokines,drugs that inhibit the differentiation of Th1 and Th17 and reduce the inflammatory factors would have therapeutic potential.ZSTK474 [2-?2-difluoromethylbenzimidazol-1-yl?-4,6-dimorpholino-1,3,5-triazine],a class I PI3K inhibitor,has been extensively studied in diseases such as cancer,autoimmune encephalomyelitis and rheumatoid arthritis,with anti-inflammatory and immunomodulatory potential.However,the possible usefulness and precise mechanisms of ZSTK474 as a treatment for Guillain-Barrésyndrome?GBS?are nclear.So far,a variety of possible treatment of GBS drugs are tested in the EAN,but the ZSTK474 for GBS immunoregulation and neuroprotective effects are not reported.Therefore,we hypothesized that ZSTK474 had a potential therapeutic effect on EAN and used ZSTK474 for drug intervention at the onset of EAN rats to collect and collate relevant data such as clinical score,inflammatory cell infiltration and demyelination degree,EMG test results and the distribution of Th1/Th17 phenotype.These were used to analyze and evaluate the therapeutic and neuroprotective effects of PI3K inhibitor,ZSTK474,on EAN rats and to explore its possible mechanism.Methods:We induced EAN in Lewis rats by subcutaneous injection of peripheral nervous system antigen P0_180-199.These EAN rats were randomly divided into control group and ZSTK474 treated group.The daily clinical score and body weight of the rats were recorded.On the 16th day post-immunization,the peak of the disease,we then compared the clinical scores and electrophysiological and histopathological changes using the Mann-Whitney U test to compare the differences between the two groups.At the peak of the disease,the degree of sciatic nerve injury was evaluated by the results of electromyography of sciatic nerve.The main evaluation indexes were motor nerve conduction velocity?MNCV?,induced compound muscle action potential amplitude?CMAP?,and latency.The sciatic nerves of EAN rats were taken and HE and fast blue staining were used to evaluate the degree of inflammatory cell infiltration and myelination,and the differentiation of Th17 cells was analyzed by immunofluorescence.The protein expression of PI3K pathway was investigated by Western blot analysis of sciatic nerve total protein.Taking the sciatic nerve while takjing the rat spleen,to obtain mononuclear cells used to analyze the proliferation of lymphocytes.The expression of various inflammatory factors was analyzed by enzyme-linked immunosorbent assay?ELISA?and polymerase chain reaction?PCR?.The differentiation of CD4⁺T cells was detected by flow cytometry.The data were analyzed by GraphPad Prism 5.0 software,and differences were considered significant at p<0.05.Results:1.ZSTK474 treated group can not only reduce the clinical score of EAN rats,but also reduce the severity of peripheral nerve injury in EAN rats compared with the control group.The ZSTK474 treated group also had reduced the area under the curve?AUC??p<0.01?.Compared with the control group,the ZSTK474 treated group had fewer inflammatory cell infiltration?p<0.01?,lighter myelination?p<0.05?.2.The results of sciatic nerve electromyography showed that ZSTK474 treated group had increased MNCV?p<0.01?,increased CMAP?p<0.05?,and shortened latency?p<0.01?.3.Flow cytometry analysis of spleen mononuclear cells showed that the number of Th1/Th17 cells in ZSTK474 treated group was significantly lower than that in control group.It indicated that ZSTK474 inhibited Th cells differentiate to Th1/Th17 cells.For P0_180-199 stimulation,compared with the control group,lymphocyte proliferation in ZSTK474 treated group were inhibited,the difference was statistically significant?p<0.01?.Without P0_180-199 stimulation,the difference was also between the two group?p<0.01?.It indicated that ZSTK474 inhibited lymphocyte proliferation.4.Immunofluorescence assay showed that the expression of Th17 cells in the ZSTK474 treated group was lower compared with the control group?p<0.01?.Western blot analysis indicated that the expression of p-STAT3 in the ZSTK474treated group decreased?p<0.01?.5.ELISA results showed that compared with the control group,proinflammatory cytokines?IL-1?,IL-1?,IFN-?and TNF-??in ZSTK474 treated group were significantly decreased at the protein level,but IL-2,IL-4,IL-6,IL-10,IL-12,IL-13,GM-CSF and RANTES were not significantly different.The same results were obtained at the translation level,and IFN-?and TNF-?were significantly decreased in the treatment group.IL-17 and IL-23 were also significantly reduced in the treatment group,indicating that ZSTK474 inhibited proinflammatory cytokines.6.Western blot analysis of p-Akt,p-mTOR,p-P70S6K,and p-4E-BP1 in sciatic nerve slices of EAN rats showed that compared with the control group,p-Akt,p-MTOR,p-P70S6K,and p-4E-BP1 reduced in ZSTK474 treated group.Conclusions:Our results suggested that ZSTK474 was effective in reducing the symptoms of EAN,which involved the inhibition of PI3K/AKT/mTOR pathway.ZSTK474 affected the differentiation of Th1/Th17 cells and further reduced pro-inflammatory cytokine production.It showed anti-inflammatory,immunomodulatory and neuroprotective effects in EAN rats.Therefore,ZSTK474may serve as a candidate for the treatment of GBS.