Role Of P38MAPK In The Substance P Induced Activation Of Spinal Cord Astrocytes And In The Rat Chronic Prostate Pain Model

BackgroundChronic prostatitis (chronic prostatitis, CP) is the most common urological diseases. The causes, pathological changes and clinical symptoms of this disease were complex and diverse. Pain is the most important symptoms in patients with chronic prostatitis. Although prostatitis is generally not life-threatening, but it can seriously affect the life quality of patients. The impact of prostatitis on mental health of patients is more serious than diabetes and chronic heart failure. Due to the etiology complexity and treatment difficulties of chronic prostatitis, although a large number of basic and clinical research have been done, but so far no breakthrough has been gained. Therefore, study of the mechanism of pain is expected to open up new treatment for chronic prostatitis. Prostate pain has the characteristics of visceral referred pain, for this reason, At present, it is recognized that the continuation and generalization of prostate pain is correlated with the secondary lesions of the L5 ~ S2 spinal cord innervating prostate.It was found that the glial cells have a role in the secondary lesions of the L5 ~ S2 spinal cord in the chronic Prostatitis as well as neurons, Glial cells, widely distributed in the brain and spinal cord, were the second largest category of cells of the nervous system in addition to neurons. In recent years, it was found that transmission and modulation of pain is not only the function of neurons, but also of activated astrocytes realized by the interaction with nerve cells and the secretion of inflammatory factors.Astrocyte function is regulated by many neurohumoral factors. At present, substance P, glutamic acid and a variety of inflammatory factors has caused widespread attention. Chronic pain can affect the function of spinal cord astrocytes through the role of these transmitters and biological factors and participate in the conduction and modulation of primary spinal cord center of chronic pain. SP, as a neurotransmitter, not only transmit physical, chemical noxious stimulation from the outside to the center leading to pain, but also can be released from nerve endings to participate in immunity, inflammation and repair of local injury after receiving the stimulating signal. In the possible mechanisms study of glial cells of L5~S2 spinal cord involved in the regulation of prostate pain, signal transduction pathway study is particularly important. p38 mitogen-activated protein kinase (p38MAPK) is an important member of the family of mitogen-activated protein kinases involved in mammalian stress and inflammatory activation.p38MAPK signaling pathway, considered as the intersection point and common pathway of the cell information transmission, involved in a variety of physiological processes such as cell growth and cellular function closely related to the regulation and control of inflammation and stress response. cAMP response element binding protein (CREB), involved in the regulation of gene expression of a variety of biological molecules, belongs to basic amino acid leucine zipper (bZIP) transcription factor family. Nuclear factor-kappa b ( NF-kB), regulating variety of inflammatory and immune gene expression, is an important factor in transcriptional regulation. There is evidence that these factors play a role in the pain mechanisms. Research on the biological characteristics of these factors and the regulation mechanism of glial cell activation of spinal cord in the chronic prostate pain contributes to a better understanding of the pathogenesis of chronic prostate pain and the promotion of effective development of anti-chronic prostate pain drug.Aim1. To observe the changes of p38MAPK of dorsal root ganglia (DRG) in L5-S2 of spinal cord and during the activation of cultured astrocytes in vitro. To observed the relationship between the changes of inflammatory factors and p38MAPK.2,To investigate the role of CREB and NF-KB in the p38MAPK-induced activation of spinal cord astrocytes, p38MAPK signal transduction pathway in the activation of spinal cord astrocytes cell and the role in the chronic prostate pain of rats.MethodsThe first part: The role of p38MAPK in the P material stimulation induced activation of spinal cord astrocytes in vitroAastrocytes, cultured from spinal cord of SPF rat and identified by immunofluorescence method with GFAP antibody, were grouped into the control group, the SP stimulus group(SP group) and the SB203580 interrupt p38MAPK group ( interrupt group)in which SP (10-7mol/L) and SB203580 (10μmol/L) were added to the supernatant for 12h. The WB method, RT-PCR method and ELISA method were used to determine the changes of p38MAPK, phosphorylated p38MAPK, GFAP mRNA, TNF-α, NO and NOS of astrocytes or supernatant at 1h,3h,12h,24h and 36h respectively.The second part: The role of p38MAPK in the activation of astrocytes of dorsal root ganglion cells in the spinal cord of rats with chronic prostatitisForty-five male SD rats were divided into the normal group (5 rats), the prostatitis group (10 rats ) and the chronic prostate pain treated with SB203580 group (the SB203580 group, 30 rats). In the SB203580 group, complete Freund's adjuvant prostate was injected and intrathecal administration of SB203580 every 5 rats with 0.5μg/10μl,2.5μg/10μl and 12.5μg/10μl were applied respectively. After 5 and 10 days, Western blot and ELISA method were used to determine the changes of p-p38 (phosphorylated p38MAPK) , GFAP, TNF-α, NO and iNOS in the posterior horn of the L5~S2 spinal cord.The third part: The role of CREB and NF-KB in the p38MAPK-induced activation of spinal cord astrocytesAastrocytes, cultured from spinal cord of SPF rat, were grouped into the normal group, the SP stimulus group(SP group), the SP stimulus + SB203580 interrupt group(SP+SB group), the SP stimulus + PD98059 interrupt group(SP+PD group) and the SP stimulus + SN50 interrupt group (SP+SN group)in which SP (10-7mol/L), SB203580 (10μmol/L), PD98059 (10μmol/L) and SN50(10μmol/L) were added to the supernatant for 12h. The WB method, immunofluorescence method and ELISA method were used to determine the changes of p-p38, p-CREB, NF-KBp65, GFAP, TNF-α, IL-1βof astrocytes or supernatant at 12h and 24h.Main results and conclusoionsThe first part: The GFAP positive rate of the cultured cells was higher than 95%. p38MAPK level in the SP group did not have any change, whereas p-p38 level increased significantly after 1h and GFAP mRNA, NO, NOS and TNF-a level increased after 3h. When p38MAPK pathway was inhibited by SB203580 in the SP+SB group, GFAP mRNA, NO, NOS and TNF-a was significantly reduced compared with those in the SP group. The level of p38MAPK, p-p38, GFAP mRNA, NO, NOS and TNF-a in the SB group did not have changes compared with those in the normal group. These results indicate that p38MAPK signal pathway contributes to P substance induced activaton of spinal cord astrocytes according to inflammatory factors attenuation after p38MAPK signal pathway interruption by SB203580.The second part: p-p38, GFAP, TNF-α, NO and iNOS of the prostatitis group increased significantly at the 5th days and 10th day. while these index were significantly reduced compared with those in the SB203580 group. The chronic prostatitis can result in the activation of p38MAPK and more expression of GFAP, TNF-αand iNOS in L5~S2 spinal cord, suggesting the secondary inflammation of L5~S2 spinal cord which is probably correlated with the maintenance and enlargement of prostate pain.The third part: p-p38, p-CREB, NF-KBp65 level in the SP group increased significantly. In the same time, GFAP, TNF-a and IL-1βlevel increased significantly too. When p38MAPK pathway was inhibited by SB203580 in the SP+SB group, p-p38, p-CREB, NF-KBp65 level and GFAP, TNF-a and IL-1βwas significantly reduced compared with those in the SP group. In the SP+PD group, p-CREB level was significantly reduced compared with those in the SP group. In the SP+SN group, NF-KBp65 level was significantly reduced compared with those in the SP group. Astrocytes from spinal cord were significant activated after stimulated by SP in vitro , Inflammatory factors levels from glial cells were significantly increased through CREB and NF-KB signaling pathways after p38MAPK activation.