Protectiveness Of Rapamycin On Spinal Cord Injuries Of Rats And Its Relative Mechanisms

The pathological process of Spinal Cord Injury (SCI) comprises primary injuryand secondary injury, with the former an irreversible injury in direct response toviolence, and the latter a series of physiological and biochemical reactions such asischemia, edema, electrolyte disturbances which follow the primary injury. Bothhypoxia-ischemia and inflammation can cause the production and release of ReactiveOxygen Species (ROS), and the massive accumulation of ROS leads to the oxidativeinjury of local tissues, as well as the apoptosis and necrosis of neurons. Therefore, ithas long been a research focus to find effective drugs for the prevention of secondaryapoptosis of spinal cord neurons.Rapamycin (Rap) has been used as a postoperative anti-rejection drug for renaltransplantation, which is an efficient immunosuppressant. It has been latelydiscovered that Rap also plays the role of upregulating autophagy. Research oninjuries of CNS show that the role of Rap may vary across animal species and models.It is an open question whether Rap can have neuroprotective effects on SCI.Through establishing the SCI model of rats, this study observed whether Rap hasneuroprotective efffects on the injured spinal cord. By virtue of PC12cellular modelwith H2O2oxidative injuries, it is further investigated whether the protective effects ofRap on neurons are mediated by interactive mechanisms between autophagy andinflammation via the expressions of NF-kB and NLRP3. The study is divided into thefollowing two parts:PART I Neuroprotectivenss of Rap on Rats with SCIObjectives: To establish the model of rats’ SCI and observe if Rap is ofneuroprotectiveness by up-regulating autophagy, down-regulating inflammation, andresisting apoptosis. Methods: The research selected45Wistar female rat, with3of them falling intosham laminectomy operation group. The other42were used for the preparation ofspinal cord impact injury models, and then be divided into Rap therapeutic group andinjury comparison group at random. Postoperative3-day,7-day and14-day specimenwere collected for H-E dyeing, ED-1dyeing and GFAP immunofluorescent dyeing,for the purpose of observing pathological changes of SCI. Enzyme histochemistrymethod was adopted for the detection on the MPO concentration of spinal cord tissues;Western blot method for the determination on the expressions of inflammatory factorsTNF-α, IL-1β and Beclin-1; TUNEL method for the determination on the apoptoticindex of spinal cord tissues, and NF dyeing for the observation of the neuron survivalin the spinal cord tissues.Results: The model of rats with spinal cord collision injuries was successfullyestablished. The MPO of the postoperative3-day Rap group was lower than that ofthe injury group (P<0.05); The expression level of Rap inflammatory factors：IL-lβand TNF-α tested with Western blot method indicated a reduction than that of injurygroup (P<0.05), while the expression of autophagic protein Beclin-1was higher thanthat of injury group; The postoperative7-day ED-1positive microglia of Rap groupwas obviously less than that of injury group (P<0.05); With the postoperative14-dayGFAP fluorescent dyeing, a downward trend of astrocyte count in Rap group wasobserved, but the distinction between the injury group and Rap group was of nostatistical significance; With H-E dyeing hypocytosis, it was observed that the injurygroup covers more inflammatory cells at the juncture of injury area and normal spinalcord. TUNEL dyeing indicated that the number of apoptotic cells in the Rap groupshowed a significant decrease compared with that in the injury group(P<0.05), and NFdyeing indicated that the number of neuron in Rap group was much more than that inthe injury group (P<0.05).Conclusion: In the model of impact injuries on the spinal cord, delivery of Rapshows a neuroprotective effect by increasing autophagy, suppressing the activationand proliferation of microglia, decreasing the infiltration of neutrophils and lymphocytes, relieving the secondary inflammatory reactions of nervous tissues,decreasing the amount of apoptotic cells and improving the survival of neuron. Part II Protectiveness of Rap on PC12Cells with Oxidative Injuries and ItsMechanismsObjectives: To observe the protectiveness of Rap pretreatment on PC12cellswith oxidative injuries, for further discussion on its mechanisms from the perspectiveof connecting signal pathways between autophagy and inflammation.Methods: The experiment is divided into groups of: normal PC12cells；H2O2+PC12；Rap+H2O2+PC12；Rap+SN50+H2O2+PC12；3-MA+H2O2+PC12. TheH2O2injury of PC12cells was prepared into models. MTT method was adopted fordetermining the survival probability of cells in different groups; Optical microscopewas employed to observe the changes of cytomorphology; MDC dyeing was forautophagic vesicles of PC12cells in the groups; ROS dyeing for cytoactive oxygen inthe groups; ELISA method for determining the supernatant of PC12cells and theintra-cellular inflammatory factors：TNF-α and IL-1β; Western blot method wasadopted for determining the changes of autophagic proteins of PC12cells: Beclin-1、LC3-Ⅱ, apoptotic proteins： Bax、 Cleave-caspase3; inflammatory signalingpathway:NF-kB; inflammasome: NLRP3and inflammatory factor：IL-1β in thegroups.Results:200μM H2O2was applied to process PC12cells for24hours, preparingthe oxidative injured cell model successfully. The MTT results proved that the cellularsurvival rates of Rap and Rap+SN50group were higher than that of H2O2group. Thecellular survival rate of3-MA group decreased than that of H2O2group. ROS stainingshowed that the average fluorescence intensity of H2O2and3-MA group wassignificantly higher than that of the normal group. Compared with H2O2injury group, Rap and Rap+SN50groups possessed much less ROS content. MDC dyeing resultsdemonstrated a smaller amount of autophagic vesicles in normal control group and alarger amount of autophagic vesicles in the H2O2injury group. In Rap and Rap+SN50group, the number of autophagic vesicles was much increased, with obviousstrengthened intracellular fluorescence particles. The number of autophagic vesicles in3-MA group were relatively small. ELISA assays found an obvious increase ofTNF-α、IL-1β secretion in the cellular supernatant in the H2O2injury group, while theTNF-α、IL-1β secretion in Rap and Rap+SN50group decreased. The result of theWestern blot determined that the Rap and Rap+SN50groups were higher than theH2O2group in the expression of autophagy related proteins LC3and Beclin-1, while3-MA group was lower than the H2O2group. The expression of inflammatorypathway protein NF-кB and NLRP3in the H2O2group was higher than that of Rapand Rap+SN50group. But the expression of NF-кB in the H2O2group was lower thanthat of3-MA group. The expression of Pro-apoptotic protein Bax and Cleave-caspase3in the3-MA group was higher than that of H2O2group, while the expression of Baxand Cleave-caspase3in the H2O2group were higher than that of Rap and Rap+SN50groups.Conclusion: In the model of PC12cells with H2O2oxidative injuries, Rapshows its neuroprotective effects by increasing the expression of autophagic proteinLC3and Beclin-1, reducing the release of ROS, reducing the expression of NF-kBand activation of NLRP3, decreasing the release of inflammatory factors: TNF-α、IL-1β, reducing the expression of pro-apoptotic proteins: Bax and Cleave-caspase3,and increasing the surviving number of PC12cells. In sum, the experiment of PC12cells cultured in vitro and vivo of rats providedevidence supporting that Rap is of neuroprotective effects. We have also initially investigated possible mechanisms underlying these effects. After the spinal cord isinjured, the application of Rap up-regulated the autophagic level, and reduced thedischarge of ROS. Through reducing the production and release of inflammatoryfactors with NF-кB and NLRP3signaling pathways, Rap down-regulated theapoptotic signals, thus decreasing the amount of apoptosis. Due to reduced release ofinflammatory factors of apoptosis, Rap down-regulated the activation andproliferation of peripheral inflammatory factors at the injured area, reduced theresponse intensity of local inflammation, decreased the amount of apoptosis andprotected the residual neurons.