Effects Of Tofacitinib On Microglia Activation And Its Mechanism In Spinal Cord Injury Repair

Spinal cord injury(SCI)is a serious traumatic disease of the central nervous system,which often leads to neurological dysfunction and greatly affects the prognosis and daily life of patients,and is an urgent clinical problem to be solved.Due to the irreversibility of primary injury,the key to the treatment of spinal cord injury is to reduce inflammation and neurological damage caused by secondary injury.In the complex pathology of SCI,the generation and amplification of early inflammation,especially the activation of macrophages/microglia,is related to the prognosis and recovery of SCI.Early disease intervention through anti-inflammatory drugs such as immunosuppressants has emerged as a potential biological treatment for SCI.As a major inflammatory signaling pathway,inhibitors of the JAK/STAT signaling pathway regulate the differentiation and presentation of immune cells and reduce the expression of inflammatory factors,and are now widely used in the treatment of many immune-related inflammatory diseases.The JAK/STAT pathway has been shown to be involved in the pathological process of SCI,but it is unclear whether targeting this pathway can alleviate SCI and whether its inhibitors can affect immune cells in the nervous system and thus have an impact on neural structures.In this study,we investigated the therapeutic effects of the pan-JAK inhibitor Tofacitinib(TOF)on SCI in rats at the early stage of SCI,as well as its effects on microglia activation and neural structure by clarifying the activation time and cellular localization of the JAK/STAT pathway in SCI.The mechanism of action of Tofacitinib was further investigated through in vitro experiments.Part Ⅰ:Activation of JAK/STAT pathway after SCIObjectiveTo investigate the temporal changes of JAK/STAT pathway activation after SCI by constructing a rat SCI model,so as to lay the foundation for the administration time of subsequent experiments;to investigate the expression level of JAK/STAT pathway in different nerve cells,so as to lay the foundation for the mechanism study of subsequent experiments.MethodsMale SD rats of similar weight and age from the same batch were randomly divided into two groups:the sham-operated group(Sham group)and the spinal cord injury group(SCI group);both groups received T10 segmental laminectomy,and the SCI group also used the modified Allen’s method to construct a SCI model.Spinal cord tissues were obtained at different times after SCI,and the expression levels and temporal characteristics of JAK/STAT pathway proteins at different time points after surgery were examined by protein blotting(western blot,WB).The activation and cellular localization of JAK/STAT pathway in spinal cord tissues after SCI were also observed by immunohistochemical staining and immunofluorescence co-staining.ResultsThe SCI group rats showed spastic tail swing and lower limb fluttering after spinal cord percussion,indicating successful modeling.The phosphorylation level of STAT1 protein in rat spinal cord tissues peaked 1-2 days after the injury,and then gradually returned to normal,slightly higher than normal on day 7;the phosphorylation level of STAT3 protein peaked on day 2 after the injury,and maintained a high level for 1 week after the injury.Histological staining showed that both STAT1 and STAT3 were highly activated in neurons at the early stage of injury(1 day),and only STAT3 was displayed at 7 days after injury.At the same time,microglia also showed strong STAT3 activation.Conclusions(1)JAK/STAT signaling pathway can be activated at the early stage of SCI,and both STAT1 and STAT3 protein levels were highly expressed after SCI.(2)STAT3 and STAT1 were mainly accumulated in neurons and activated microglia after SCIPart Ⅱ:Tofacitinib inhibits inflammation through JAK/STAT pathway to promote motor function recovery after SCI in ratsObjectiveTo investigate the effect of different doses of TOF,a pan-JAK inhibitor,on the recovery of motor function in rats given at the early stage of SCI according to the activation time of JAK/STAT pathway-related proteins in the first part;to investigate the trend of changes in the level of inflammation in spinal cord tissue after SCI and the therapeutic effect of TOF on inflammation at the early stage after SCI;to investigate the effect of TOF on the activation of microglia according to the cellular localization of STAT protein activation in the first part.The effect of microglia activation was investigated.MethodsThe rats were divided into four experimental groups,including sham-operated group(Sham group),spinal cord injury group(Vehicle group),low-dose TOF treatment group(TOF-5 group)and high-dose TOF treatment group(TOF-10 group),according to the different administration and surgical methods.The TOF-5 and TOF-10 groups were given intraperitoneal injection of TOF at a dose of 5 mg/kg and 10 mg/kg,respectively,while the Sham and Vehicle groups were given saline.The BBB(Basso,Beattie,Bresnahan Scales)scale and Louisville Swim Score(LSS)were used to assess the recovery of motor function and somatic balance in each group.The spinal cord tissue supernatant was extracted after injury,and the levels of inflammatory factors in the early stages of SCI were measured by enzyme-linked immunosorbent assay(ELISA).The polarization status of microglia in spinal cord tissues was detected by immunofluorescence staining.The expression of polarization-related markers in spinal cord tissues was detected by WB.ResultsThe motor function of rats started to recover gradually on the 7th day after SCI,and the recovery rate was more obvious after TOF treatment.The BBB score and LSS score were significantly higher than those of the Veh group(P value<0.05),and the effect was better in the high concentration treatment group.The swimming and crawling experiments showed that the treated rats showed better trunk balance and limb coordination.The treatment of TOF significantly improved these pathological features,reduced the inflammatory infiltration,and improved the stenosis and depression of the spinal cord caused by the compression.After SCI,high expression of TNFα,IL-1β and IL-6 protein levels could be seen at 12 h,followed by a gradual return to baseline levels within 1 week.At 1-3d post-injury,inflammatory factor levels exhibited a significantly different downregulation after TOF treatment(P value<0.05),and the decline to the uninjured state was much shorter.Immunofluorescence staining further showed that the inhibition of inflammation by TOF was closely related to its regulation of microglia activation,reducing microglia aggregation in the injury area overall,while elevating the proportion of M1-type pro-inflammatory microglia characterized by iNOS and decreasing the proportion of M2-type anti-inflammatory microglia characterized by Arg-1,and decreasing iNOS at the protein level expression and promoted Arg-1 expression.WB results of JAK/STAT pathway proteins showed that TOF effectively reduced the levels of ST AT1 and ST AT3 phosphorylated proteins in a dose-dependent manner.Conclusions(1)TOF significantly promoted the recovery of motor function and reduced the hematoma of spinal cord tissue in rats with SCI.(2)Early application of TOF after SCI was effective in reducing the level of early inflammation in spinal cord tissues.(3)TOF inhibited the inflammatory response to SCI,possibly by inhibiting M1-type polarization of microglia and promoting M2-type polarization.(4)TOF modulates the activation of JAK/STAT pathway after SCI.Part Ⅲ:Tofacitinib inhibits inflammation through JAK/STAT pathway to promote motor function recovery after SCI in ratsObjectiveTo investigate the pathological changes of spinal cord neural structure in the subacute stage of SCI in rats,and the protective effect of TOF on neurons;to investigate the effect of TOF on neuronal apoptosis after SCI.MethodsThe rats were molded and administered in groups according to the method in Part Ⅱ,and the spinal cord tissues in the subacute and chronic stages of SCI were obtained.The neurological damage and demyelination were observed by hematoxylin-eosin(HE)staining and Luxol fast blue(LFB)staining;the formation of glial scar,histological changes of nerve fibers and axons,and neuronal apoptosis were detected by immunofluorescence co-staining after SCI.The expression of individual neural structural markers and the apoptosis level of neurons in spinal cord tissues were detected by WB means.ResultsThe spinal cord tissue showed typical pathological changes of injury healing after SCI.In the subacute stage,tissue structure fragmentation appeared in the center of the injury,and in the chronic stage,obvious tissue cavities and necrotic foci were formed,which disrupted the continuity of the spinal cord tissue.Immunofluorescence staining further showed that astrocytes around the injury area were heavily aggregated and formed glial scar,and there was extensive loss of dendrites,axons,myelin and other neural structures in the center of the injury area.TOF treatment reduced the area of the lesion,preserved more myelin and axonal structures,and promoted the penetration of dendritic fibers in the non-injured area into the injury center by inhibiting the formation of glial scar.Spinal cord impact injury resulted in a reduction and overlapping aggregation of neurons in the damaged area,interrupting the normal arrangement of neurons and causing widespread apoptosis in the center of the injury.TOF treatment allowed more neurons to survive in the injured area,significantly reduced C-CASP3 fluorescently labeled apoptotic cells,and corrected the elevated expression of C-CASP3 at the protein level after SCI,and reduced the Bcl2/Bax ratio was reduced.Conclusions(1)TOF improved the pathological morphology of the spinal cord after SCI.(2)TOF inhibited the formation of glial scar and promoted the growth of dendritic fibers.(3)TOF promoted the preservation of axons,myelin sheaths and other neural structures after SCI.(4)TOF inhibited the apoptosis of nerve cells after SCI and promoted the survival of neuronal cells.Part Ⅳ:Tofacitinib inhibits inflammation through JAK/STAT pathway to promote motor function recovery after SCI in ratsObjectiveTo investigate the toxic effects of TOF on microglia under in vitro conditions;to establish an in vitro microglia pro-inflammatory activation model to investigate the effect of TOF intervention on microglia secretion of inflammatory factors and the regulation of microglia activation phenotype.MethodsMouse primary microglia were extracted,and in vitro cell experiments were performed for primary cells and microglia line BV2,respectively.After determining the toxic dose of TOF on microglia,the cells were pretreated with different doses of TOF and subsequently induced by lipopolysaccharide(LPS)to construct an in vitro inflammation model of microglia.The phenotype of microglia was identified by flow cytometry,the expression levels of different factors were analyzed by qPCR,microglia activation was observed by immunofluorescence staining,and the expression of JAK/STAT pathway proteins was detected by WB.ResultsCCK-8 results showed that TOF was not toxic to microglia below a concentration of 100 μM.After LPS induction,microglia showed significant morphological and functional manifestations of M1 polarization,with significant increases in STA1 and STAT3 phosphorylation levels.Administration of TOF pretreatment reversed the LPS-induced amoeboid morphological changes in microglia in a dose-dependent manner and reduced the fluorescent labeling of iNOS.Flow cytometry detected a decrease in the proportion of M1-type cells(iNOS and F4/80 positive);qPCR results showed downregulation of M1-related genes iNOS,IL-6,TNF-α and IL-1β,and somewhat elevated the levels of M2-associated genes Arg-1 and CD 163;WB results showed that TOF inhibited the activation of STAT1 and STAT3 by iNOS expression and promoted the expression of Arg-1.Conclusions(1)Microglia undergo M1 polarization after LPS induction in vitro activates the JAK/STAT pathway.(2)TOF inhibits LPS-induced M1 polarization via the JAK/STAT pathway in a dose-dependent manner and promotes M2 polarization to some extent in an in vitro model of inflammation.Part Ⅴ:The protective effect of tofacitinib on neuronal cells under in vitro conditionsObjectiveTo investigate the toxic effect of TOF on microglia under in vitro conditions;to establish an in vitro microglia-neuronal cell co-culture model to investigate the protective effect of TOF on neuronal cells under inflammatory environment.MethodsThe primary mouse neuron cells were extracted,and the primary cells and the mouse hippocampal neuron cell line HT22 were tested in vitro.After determining the toxic dose of TOF to neurons by CCK-8 method,the co-culture model of inflammatory BV2 cells and neurons was constructed.Subsequently,flow cytometry(Annexin V-FITC/PI)was used to analyze the early apoptosis of neurons,and WB was used to detect the expression of apoptosis-related proteins in neurons,so as to analyze the protective effect of TOF on neurons under inflammatory conditions.ResultsCCK-8 results show that TOF is 100 μ There is no toxicity to neurons below the concentration of M.Under the inflammatory co-culture condition,LPS-induced BV2 cells inhibited the proliferation and survival of neurons and destroyed the normal axonal structure.The pretreatment of neurons by TOF reversed this phenomenon,reduced the proportion of early apoptotic cells and decreased the expression of apoptosis-related proteins.Conclusions(1)In vitro,activated microglia can produce toxic effects on neurons and promote apoptosis of neurons;(2)TOF promotes the survival of neurons in inflammatory state in a dose-dependent manner,and inhibits the early apoptosis of neurons.