| PART1. THE EXPERIMENTAL STUDY OF LIPOPOLYSACC-HARIDE PRECONDITIONING ON THE REPAIR OF SPINALCORD INJURY IN RATSObjective: The experimental model of traumatic SCI in rats werebuilt by the modified Allen’s and NYU impactor methods, respectively. Toinvestigate the effects of antiapoptosis, functional recovery and theexpression of transcription factor Nrf2after low-dose LPS-inducedimmune cross-tolerance, hoping to provide experimental and theoreticalbasis for LPS-induced immune tolerance in central nervous system and anew target of prevention and therapy for SCI.Methods:(1) One hundred and thirty-two adult SD rats wererandomly divided into three groups: sham operation (n=12), model (n=60),LPS preconditioning (n=60). The model of ASCI was built by the modifiedAllen’s method, and was assessed by the BBB scores at1,3,5and7d afterthe operation. The injured spinal cord tissue samples were collected at6,12 h, and1,3,5and7d after the operation. The pathological changes of ratspinal cord were observed by HE staining, the Nissl body in neurons wasobserved by Nissl staining, the expressions of Nrf2and caspase-3weredetected by immunhistochemistry, and the Nrf2protein level was detectedby Western blotting.(2) Seventy-two adult female SD rats were randomlyassigned to one of four groups: sham injury, control, LPS preconditioningand MP groups, with18rats in each group. The model of TSCI was builtby the NYU impactor method, and was assessed by the BBB scores at6,12,24,48and72h after injury. The animals were euthanized at72hpostinjury. Neuropathologic changes were assessed by HE and Nisslstaining. SCI-induced apoptosis were detected by TUNEL staining andtransmission electron microscopy (TEM). Nrf2, caspase-3, cleavedcaspase-3, Bax and Bcl-2were examined with immunhistochemistry orWestern blotting.Results:(1) The rat BBB scores decreased significantly in both themodel group and LPS preconditioning group at1d and3d after operationand was higher in LPS preconditioning group than in the model group at5d and7d after operation (P＜0.05). The injured spinal cord by HE andNissl staining showed an extensive focal hemorrhage, large quantity oferythrocyte diapedesis, severe interstitial edema, karyopyknosis,significantly decreased number of neurons, samall cell volume and blurringand debris of Nissl bodies in the model group and LPS preconditioning group at6h,12h and1d after operation. Low Nrf2and trace caspase-3expressions were detected in sham group. As compared with those of themodel group, the morphological injury was significantly alleviated, theNrf2protein expression was significantly enhanced (P＜0.01, P＜0.05) andthe number of caspase-3positive cells was significantly decreased (P＜0.01, P＜0.05) in LPS preconditioning group at1,3,5and7d afteroperation.(2) Compared with the control group, the BBB scores in LPSpreconditioning group were significantly improved at24,48and72h afterinjury (P＜0.05). There were no statistical differences in BBB scoresbetween LPS preconditioning group and MP group at every time point afterinjury. Compared with the control and MP groups, the morphologicalchanges of spinal cord tissues in LPS preconditioning group weresignificantly improved at72h after injury, while transmission electronmicroscope and TUNEL staining showed the apoptosis of nerve cells inLPS preconditioning group were significantly reduced (P＜0.01).Compared with the control and MP groups, IHC and Western blottingshowed that the expressions of apoptotic proteins caspase-3, cleavedcaspase-3and Bax in LPS preconditioning group were significantlyreduced but the expressions of transcription factor Nrf2and antiapoptoticmarker Bcl-2in LPS preconditioning group were significantly upregulatedat72h after SCI.Conclusion: Our results demonstrate that low-dose LPS preconditioning can inhibit the accumulation of inflammatory cells, theproliferation of glial cells and neuroapoptosis in injuried spinal cord, whichcould be one of the underlying mechanisms of low-dose LPS to promotenerve regeneration. Low-dose LPS preconditioning can provide protectiveeffects against TSCI in rats by activating transcription factor Nrf2andregulating caspase-3, cleaved caspase-3, Bax and Bcl-2. Therefore, theinhibition of nerve cells apoptosis may be another related mechanism toLPS-induced protective tolerance in central nervous system.PART2. SiRNA TRANSFECTION ON SPINAL CORDTISSUES IN RATS AND THE EFFECT OF SiRNA-MEDIATEDKNOCKDOWN OF ERK OR NRF2Objective: To evaluate the effect of siRNA transfection on spinalcord tissues in rats by local intraspinal injection, and the effect ofsiRNA-mediated knockdown of ERK or Nrf2by RT-PCR and Westernblotting, hoping to prepare materials for the followed experiments.Methods: Adult female SD rats were fed for3days in theexperimental animal center. Then, the diluted solution that containingfluorescence lentiviral vector was injected0.8mm into the dorsal columnof the spinal cord at T10by microsyringe. To observe the effect of siRNAtransfection at different points by fluorescence microscopy. ERK-siRNA and Nrf2-siRNA were transfected into local spinal cord tissues in rats,respectively, and RT-PCR and Western blotting were employed to detectthe change of ERK and Nrf2mRNA and protein expressions7dayspost-transfection.Results: Compared with the sham operation group, the expression offluorescence of each time point in model group was enhanced afterlentiviral transfected, but the effect of transfection was more remarkable at3and7d. The expression of fluorescence in model group reached the peakafter transfected7days, and the spinal cord tissues expressed fluorescencein a time-dependent manner. RT-PCR results showed that after transfected7days, in the sham operation and the model groups ERK mRNAexpression was significantly reduced (P＜0.01, P＜0.05), in the two groupsNrf2mRNA expression was significantly reduced (P＜0.01). Westernblotting results showed that after transfected7days, in the sham operationand the model groups ERK protein expression was significantly reduced (P＜0.01, P＜0.05), in the two groups Nrf2protein expression wassignificantly reduced (P＜0.01).Conclusion: SiRNA could be transfected into the spinal cord tisues inrats by local intraspinal injection. ERK and Nrf2gene expression of spinalcord tissues can be effectively inhibited by RNA interference. PART3. THE ANTIAPOPTOSIS MECHANISM RESEARCHOF LIPOPOLYSACCHARIDE PRECONDITIONING ONSPINAL CORD INJURY IN RATSObjective: To investigate antiapoptosis molecular mechanism andtarget of LPS preconditioning on SCI, hoping to provide experiment basisfor clinical practice and therapy.Methods:(1) Forty-eight adult female SD rats were randomlyassigned to one of four groups: control (group a), LPS+control (group b),ERK1/2interference (group c) and LPS+ERK1/2interference (group d)groups, with12rats in each group. The model of TSCI was built by theNYU impactor method. The animals were euthanized at72h postinjury.SCI-induced apoptosis were observed by TUNEL staining and TEM. Theprotein expressions of total Nrf2, HO-1, NQO1and GCLC were examinedwith immunhistochemistry and Western blotting. The protein expressionsof Nrf2in cytoplasm and nucleus were respectively detected by Westernblotting.(2) Forty-eight adult female SD rats were randomly assigned toone of four groups: control (group e), LPS+control (group f), Nrf2interference (group g) and LPS+Nrf2interference (group h) groups, with12rats in each group. The model of TSCI was built by the NYU impactormethod. The animals were euthanized at72h postinjury. SCI-inducedapoptosis were observed by TUNEL staining and TEM. The protein expressions of HO-1, NQO1and GCLC were examined withimmunhistochemistry and Western blotting.Results:(1) In the a and c groups, TEM showed that a large numberof apoptotic neurons exhibited abnormal morphology with volume atrophy,vacuolized cytoplasm, karyotin margination and discontinuous nuclearmembranes. Neurons in the b group showed less abnormal morphology andclear nuclear structure. Compared with the a and d groups, TUNEL stainingshowed quantity of TUNEL-positive cells in the b group was significantlyreduced (P＜0.01). Compared with the d group, IHC showed that thenumber of Nrf2, HO-1and NQO1positive cells in the b group weresignificantly increased (P＜0.01), but there was no significantly differencein quantity of GCLC positive cells between b group and d group.Compared with the a group, the number of Nrf2, HO-1, NQO1and GCLCpositive cells in the b group were significantly increased (P＜0.01, P＜0.05). The protein expressions of total Nrf2, Nrf2in cytoplasm, Nrf2innucleus, HO-1and NQO1in the b group were significantly increased thanthat in d group (P＜0.01), but there were no significantly differences inGCLC protein expressions between b group and d group. Compared withthe a group, Western blotting showed that the expressions of all proteins inthe b group were significantly enhanced (P＜0.01, P＜0.05).(2) In the e, gand h groups, TEM and TUNEL staining showed that a large number ofapoptotic nerve cells was found but quantity of apoptotic nerve cells in the f group was significantly decreased (P＜0.01). Nerve cells in the f groupshowed more clear morphology and structure. Compared with the e and hgroups, IHC showed that the number of HO-1, NQO1and GCLC positivecells in the f group were significantly increased (P＜0.01). The proteinexpressions of HO-1, NQO1and GCLC in the f group were significantlyenhanced than that in e and h groups (P＜0.01). Compared with the e group,Western blotting showed that the protein expressions of HO-1and NQO1in the g group were significantly reduced (P＜0.01, P＜0.05).Conclusion: Low-dose LPS preconditioning triggeredERK1/2-Nrf2/ARE signaling to induce the neuroprotection of spinal cordtissues in a cross-tolerance manner. The pathway can mediate antioxidantfactor expressions in local spinal cord, reduce secondary apoptosis andinflammatory responses, protect neurons and promote functional recovery. |
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