Effects Of Protein Kinase C On The Neuronal Apoptosis And Bax Protein Expression Of Spinal Cord Of Rats With Inflammatory Pain

Objective: Protein kinase C (PKC) is a protein kinase family including serine kinase and threonine kinase which can be activated by Ca²⁺ in the cytoplasm and plays an important role in the cell signal transduction. PKC locates in many tissues of body including central neuronal system and plays a key role in regulation of neuronal excitability, cell sigal transduction, neurontransmitters release and synaptic plasticity. PKC is unactivated in the cytoplasm in the resting circumstance, and can be activated by the activator then it translocates to the plasmalemma playing its role.The previous studies of our labotary proved that formalin inflammatory pain could induce the activation of PKC in spinal cord of rats. Also we proved that the neuronal apoptosis in spinal cord of rats could be induced by formalin inflammatory pain. It was reported that the activation of PKC participated in nociceptive information input and in hyperalgesia in the spinal cord. And it is unknown whether PKC participate in neuronal apoptosis in spinal cord of rats with formalin inflammatory pain.Bax protein plays an important role in regulating apoptosis and promoting apoptosis. Bax protein locates in cytoplasm and widely distributes in every tissues. And it is unclear whether or not PKC effects on the neuronal apoptosis by influencing the expression of Bax protein.Therefore, using Flow cytometry, we observed the effect on the ratio of neuronal apoptosis of rats with inflammatory pain by intrathecal injection of PKC selective inhibitor chelerythrine chloride(CH) in this study and also observed the effect of PKC on the expressin of Bax protein in spinal dorsal horn of rats with Inflammatory Pain by immunohistochemistry to study the effect of PKC participating in neuronal apoptosis and the mechanisms in spinal cord of rats with formalin inflammatory pain.1 Effect of PKC on the neuronal apoptosis in spinal neurons of rats with formalin imflammatory painTwenty five male SD rats (280±20 in weight) were divided randomly into 5 groups: control (control) group, formalin (F) group, formalin+DMSO(F+DMSO) group, formalin +CH(F+CH) group and PMA group. There were five rats in each group. Rats in control group were directly sacrificed without other treatments. After rats of F group were subcutaneously injected with formalin into the ventral surface of the right hind paw, and they were sacrificed after 3 day. The F+DMSO group and F+CH group were intrathecal injection of DMSO and CH solution 10 10μl 15 minutes prior to formalin. After formalin injected 3 day rats were sacrificed. Normal animals were intrathecal injection of PMA and were sacrificed after 3 day. Flow cytometry was used to assay the ratio of neuronal apoptosis of L4-5 segment of rat spinal cord.The results were as follows: the ratio of neuronal apoptosis in L₍4-5 segment of the rat's spinal cord was very low in control group. Comparing with control group, the ratio of neuronal apoptosis of rat's spinal cord in F group significantly increased(P<0.01). Comparing with F group, the ratio of neuronal apoptosis in spinal cord of rats in F+DMSO group was no significant difference(P>0.05), indicating there was no influence on the ratio of neuronal apoptosis of rat's in spinal cord intrathecal injection of DMSO to rats with formalin inflammatory pain. Comparing with F group, the ratio of neuronal apoptosis in spinal cord of rats in F+CH group was significantly decreased(P<0.01). Comparing with control group, the ratio of neuronal apoptosis of rat's spinal cord in PMA group significantly increased(P<0.01).The results showed that intrathecal injection of CH could inhibit the apoptosis of spinal neurons of rats with formalin inflammatory pain. PKC has a promoting role in the apoptosis process of spinal neurons of rats with formalin inflammatory pain.2 Formalin inflammatory pain induced the expression of Bax protein in spinal horn neurons of rats and the effect of PKC on the expression of Bax protein2.1 Formalin inflammatory pain induced an upregulation expession of Bax protein in spinal cord dorsal horn neurons of ratsTwenty five male SD rats (280±20 in weight) were divided randomly into 5 groups: control group, formalin 12h group, formalin 1d group, formalin 2d group, formalin 3d. There were five rats in each group. Rats in control group were directly sacrificed without other treatments. Rats of formalin groups were subcutaneously injected with formalin into the ventral surface of the right hind paw to induce periphery inflammatory pain. L4-5 segment of spinal cord was dissected out at the designed ending time point in formalin group. The immunohistochemistry was used to observe the expression of Bax protein in the lamina I-II of the L4-5 spinal dorsal horn neurons.The results were as follows: after rats were injected formalin into the ventral surface of the right hind paw, and they immediately manifested a biphasic pain reaction, including biting, flinched and scratch the wound paw. The first one appeared right after the injectin and lasted for several minutes. Then there is a silent period. Then the second one appeared. The flinches lasted about 1 hour.The results of immunohistochemical staining showed that there was rare immunoreactive positive cells of Bax protein in the lamina I-II of the L4-5 spinal dorsal horn of rats in control group. Comparing with control group, the area and the average optical density of Bax immunoreactive cells in the lamina I-II of the L4-5 spinal dorsal horn of rats in F group increased(P<0.01), which manifestationed that immunorective cells stained strongly; And the increase in area and stained degree of Bax immunoreactive cells was the most obviosly at 1d after formalin injection; The area and the average optical density of Bax of F2d began to decrease, but they were higher than rats in control group and F3d group(P<0.01).The results suggested that formalin inflammatory pain could induce the expression of Bax protein in spinal cord dorsal horn of rats. And the upregulation of Bax protein expression was the most obvious at 1d after formalin injection.2.2 The cffect of protein kinase C on the expression of apoptosis-associated protein Bax in spinal horn neurons Twenty five male SD rats (280±20 in weight) were divided randomly into 5 groups: control (control) group, formalin (F) group, formalin+DMSO(F+DMSO) group, formalin +CH(F+CH) group and PMA group. There were five rats in each group. Rats in control group were directly sacrificed without other treatments. Rats in F group were subcutaneously injected with formalin into the ventral surface of the right hind paw, and were sacrificed after 1day. The F+DMSO group and F+CH group were intrathecal injection of DMSO and CH solution 10μl 15 minutes prior to formalin. Animals were sacificed after formalin was injected 1 day. Rats were observe the flinch after normal animals were intrathecal injection of PMA and animals were sacrificed after 1day. The immunohistochemistry was used to observe the expression of protein Bax in the lamina I-II of the L4-5 spinal dorsal horn neurons.The results were as follows: Comparing with control group, the expression of protein Bax in the lamina I-II of the L4-5 spinal dorsal horn neurons in F group rats increased(P<0.01), which manifestationed that both of area and the average density of Bax immunoreactive cells were increased. Comparing with F group, the area and the average density of Bax immunoreactive positive cells were no significant difference in the rats of intrathecal injection of DMSO(P>0.05), indicating there was no influence on the expression of Bax protein of rats intrathecal injection of DMSO to rats with formalin inflammatory pain. Comparing with F group, the area of Bax immunoreactive cells in the lamina I-II of the L4-5 spinal dorsal horn neurons in F+CH group were significantly decreased(P<0.01). And the positive cells were stained obviously lightly(P<0.01). Comparing with control group, the area and the average density of Bax immunoreactive cells were obviously increased in the lamina I-II of the L4-5 spinal dorsal horn neurons in PMA group(P<0.01).The results suggested that the expression of Bax protein induced by nociceptive input in spinal dorsal horn neurons of rats were inhibited by intrathecal injection of CH; and intrathecal injection of PMA could induce an upregulation of expression of Bax protein.Conclusion: 1 This study confirmed that intrathecal injection of PKC inhibitor CH could inhibit the neuronal apoptosis process in spinal cord of rats with formalin inflammatory pain. The activation of PKC could promote the neuronal apoptosis in spinal cord of normal rats.2 It enhanced the expression of Bax protein in spinal dorsal horn of rats by injecting formalin into the ventral surface of the paw. And it expressed most after formalin was injeced for 1 day. Intrathecal injection PMA to normal rats could induced the nociceptive response of rats and the expression of Bax protein in spinal horn. It could inhibit the expresstion of Bax protein in the spinal cord dorsal horn of rats by intrathecal injection of PKC inhibitor CH prior to formalin.3 These results suggested that PKC promote the neuronal apoptosis that induced by formalin inflammatory pain. And it was one of the mechanisms that PKC influences the expression of Bax.