Investigating Amlexanox Inhibits Th1 And Th17 Cellular Responses And Its Neuroprotective Effects In EAE Mice

Objective: In this experiment,EAE mice were used as the animal model,drug intervention was performed by using ALX.Evaluated the incidence and onset time in each group of mice.The effects of ALX on the pathological aspects of EAE mice were studied by HE staining,LFB staining and transmission electron microscopy.To study the effects of ALX on inflammatory infiltration and demyelination in EAE mice.The ratio of Th1 and Th17 cells in spleen were determined by flow cytometry.Real-time PCR and ELISA were used to detect the expression of Th1 and Th17 related inflammatory cytokines in spleen from the levels of transcription and protein expression,respectively.To study the effects of ALX on peripheral Th1 and Th17 cell responses.Investigating protection effects of ALX in EAE.Provide a new theoretical basis for clinical drug treatment of multiple sclerosis.Methods:Thirty female C57BL/6 mice weighing 18 g to 20 g and 8-10 weeks were randomly assigned to control,EAE and ALX groups.EAE group,ALX group established the mouse model of EAE by using MOG35-55 which was assisted by Mycobacterium tuberculosis,Complete Freund’s Adjuvant and Pertussis Toxin.The control group were not immuned.Since the day of immunization,the ALX group mice were orally given ALX solution 50mg/kg twice a day(8:00 and 16:00);The EAE group and control group mice were orally given the same amount of CMC at the same time.From day 1,the mice neurological function was evaluated with the method of double blind methodby the two observers at the same time as above day by day(8:00 and 16:00)and recorded the scores by using Kono’5 points.Thirty-six of the same mice were randomly divided into EAE group and ALX group.Give the same treatment as above.23 days after immunization(the peak of onset time),the mice in EAE and ALX groups were sacrificed and obtained.The lumbar enlargement of spinal cord were taken and studied by HE staining,LFB staining and transmission electron microscopy.At the same time as above,fresh tissue were taken from each group of mice.Flow cytometry was used to detect the ratio of Th1 and Th17 cells in spleen.The levels of IFN-γ and IL-17 in cultured supernatants of spleen cells were detected by ELISA.The mRNA transcriptional levels of cytokines IFN-γ and IL-17 were detected by rtPCR.Results:1.The incidence and Onset time of mice in each groupThe incidence of EAE group was 100%.The disease incidence of ALX group was 80%.The control group did not develop the disease.The ALX group delayed onset compared with EAE group,and the difference was statistically significant.(P<0.001).2.HE stainingEAE group mice showed a large number of inflammatory cells infiltrating the spinal cord.Most occurred at the junction of gray and white matter and the anterior cord of the spinal cord.The score of inflammatory lesions in spinal cord tissue of ALX group were decreased compared with the mouse of EAE group,and the difference was statistically significant.(P<0.05).3.LFB stainingEAE group of mice can be observed different size of patch demyelinating region in the spinal cord.It was often accompanied by a large part of the infiltration of inflammatory cells.The ALX group with a relatively mild degree of demyelination.The lesion area became lighter in color,and the demyelination score display difference was statistically significant.(P<0.001).4.Transmission electron microscope scanningThere were leukoaraiosis,loose myelin,myelin disintegration in the lumbar enlargement of EAE mouse.In the ALX group,the structure of lumbar enlargement was relatively complete and the concentric circle was arranged into the rules.5.The ratio of Th1 and Th17 cellsCompared with EAE group,the ratio of Th1 cells was more significantly decreased in ALX group(P <0.05).Compared with EAE group,the ratio of Th17 cells was more significantly decreased in ALX group(P <0.001).6.The concentration of IFN-γ and IL-17 in cultured supernatants of spleen cellsCompared with EAE group,the IFN-γ concentration in ALX group was more significantly decreased(P<0.001).Compared with EAE group,the concentration of IL-17 in ALX group was more significantly decreased(P <0.05).7.The mRNA ]transcriptional levels of IFN-γand IL-17 of the spleen tissue and spinal cord in each groupCompared with EAE group,the levels of IFN-γmRNA in spleen in ALX group were more significantly decreased(P <0.01).Compared with EAE group,the levels of IL-17 mRNA in spleen in ALX group were more significantly decreased(P <0.001).Conclusions:1.ALX can delay the onset of EAE mice,reduce neurological scores in EAE mic and play a role in alleviating the disease.2.ALX can significantly reduce inflammatory cell infiltration and the demyelination of spinal cord in EAE mice.3.ALX can decrease the ratio of Th1 and Th17 cells and the mRNA and protein expression of periphery inflammatory cell IFN-γ and IL-17.4.ALX may play a neuroprotective role in EAE mice by inhibiting the inflammatory response of Th1 and Th17 cells.