【作者】 刘超；

【导师】 严军；

【作者基本信息】 苏州大学 ， 骨外科（专业学位）， 2024， 博士

【摘要】 研究的背景与目的脊髓损伤（Spinal cord injury,SCI）是一种破坏性神经系统疾病,会导致严重的运动、感觉及自主神经功能障碍,发病率居高不下,给社会带来严重的经济负担,目前仍缺乏有效的治疗手段。复杂的病理生理机制是导致SCI治疗失败的主要原因。研究表明,细胞焦亡、铁死亡及线粒体自噬在SCI的病理过程中起着重要作用,而抑制细胞死亡,促进线粒体自噬可能成为治疗SCI的新策略。硫化氢已被证明在SCI中具有神经保护作用,但详细作用机制并不明确,其可能是通过调节内源性硫化氢（Hydrogen sulfide,H₂S）的合成发挥作用,但还有待进一步探索;其次,在SCI中,H₂S是否可以通过调节细胞焦亡、铁死亡及线粒体自噬发挥作用也鲜有报道;而且,H₂S作为气体信号分子,具有强挥发性,且一过性挥发可致局部高浓度产生细胞毒性,也阻碍了其应用前景,因此,如能找到合适的载体使其缓慢释放,将更好地发挥其神经保护作用。丝素蛋白水凝胶（Silk Fibroin Hydrogels,SF-Gel）作为新型天然蛋白材料,在组织工程和再生医学领域中被广泛应用,它在与一些特定药物相结合后可以明显延缓药物的半衰期,而H₂S在体内主要以HS-离子形式存在,可能与丝素蛋白水凝胶天然结构相结合,达到一个低剂量缓慢释放效果,从而更好地发挥作用。在本研究中,我们旨在构建H₂S缓释的丝素蛋白水凝胶,应用于大鼠SCI模型,观察其神经功能保护作用;探讨外源性H₂S在SCI中对细胞焦亡、铁死亡及线粒体自噬的调节作用,初步探索线粒体自噬与细胞焦亡之间的联系,以期为SCI的临床治疗提供新的思路。研究方法第一部分:选用Na HS作为H₂S供体,通过物理溶解与丝素蛋白水凝胶混合,制得可注射包载硫化氢的丝素蛋白水凝胶（H2S@SF）,利用发射扫描电子显微镜观察水凝胶及包载H₂S后水凝胶形态;Xplo RA激光拉曼光谱仪对Na HS水溶液、SF水凝胶及包载不同浓度H₂S的丝素蛋白水凝胶进行光谱检测;微型硫化物离子电极检测H₂S释放情况;CCK-8实验对水凝胶及包载H₂S水凝胶进行细胞毒性检测。建立大鼠SCI模型,分别用Na HS及H2S@SF进行腹腔注射干预,然后将大鼠随机分为对照组（Sham组）、SCI组、Na HS组及H2S@SF组。运用尼氏染色、FJB染色及免疫荧光染色检测各组神经元变性和生存情况;Westen blot及免疫荧光染色检测各组突触素SYP的表达情况,评估轴突功能;运用BBB评分及足迹实验评估水凝胶包载H₂S后对大鼠运动功能恢复情况。第二部分:将大鼠随机分为Sham组及SCI后不同时间点组（1h、6h、12h、1d、2d、3d、7d）,Westen blot检测各组大鼠脊髓组织中焦亡相关蛋白（Caspase 1、GSDMD、ASC）的表达情况,分析其时序性特点;免疫荧光染色检测焦亡相关蛋白细胞定位情况。按第一部分方法将大鼠随机分为Sham组、SCI组、Na HS组及H2S@SF组,Westen blot检测各组焦亡相关蛋白（Caspase 1、GSDMD、ASC）及内源性H₂S合成酶相关蛋白（CBS、3-MST、CSE）的表达情况,免疫荧光染色检测CBS细胞定位情况;微型硫化物离子电极检测各组大鼠脊髓组织、血液及脑脊液中H₂S的相对含量,评估外源性H₂S的调控作用及水凝胶包载H₂S缓释后的效果。然后分别用焦亡抑制剂AC-YVAD-CMK和CBS抑制剂AOAA进行干预,Westen blot检测焦亡相关蛋白及内源性H₂S合成酶相关蛋白的表达;探索H₂S在细胞焦亡中的调控机制在此基础上,Westen blot检测Sham组、SCI组、Na HS组及H2S@SF组的铁死亡相关蛋白（GPX4、COX2、x CT）的表达情况;免疫荧光染色检测铁死亡核心蛋白GPX4细胞定位情况,评估外源性H₂S对铁死亡的调控作用及水凝胶包载H₂S缓释后的效果。第三部分:在上述基础上,Westen blot检测Sham组、SCI组、Na HS组及H2S@SF组的线粒体自噬相关蛋白（LC3、PINK1、Parkin、P62）的表达;免疫荧光染色检测PINK1细胞定位情况;评估外源性H₂S对线粒体自噬调控作用及水凝胶包载H₂S缓释后的效果。然后Westen blot检测线粒体自噬-溶酶体相关蛋白Rab7在Sham组及SCI后不同时间点组（1h、6h、12h、1d、2d、3d、7d）的表达,探索其时序性特点,免疫组化进一步验证;免疫荧光染色检测Rab7细胞定位情况;再用焦亡抑制剂AC-YVAD-CMK进行干预后,Westen blot检测Rab7表达情况;探索线粒体自噬与细胞焦亡之间的联系。研究结果第一部分:电子显微镜显示H2S@SF具有开放和互连的多孔形态特征;拉曼光谱显示所有凝胶组均在1662cm-1处呈现出归属于酰胺Ⅰ的丝素蛋白特征峰,表明SF水凝胶具有β-折叠结构,分子构象稳定;2571 cm-1处呈现出归属于-S-H-键的特征峰,且随着凝胶中Na HS搭载浓度的增加特征峰越发明显,说明SF水凝胶对H₂S实现了有效搭载。微型硫化物离子电极结果显示,Na HS溶液中H₂S在2h释放达90%以上;4h基本释放殆尽,而H2S@SF中H₂S在2h释放约60%,72h后释放殆尽,说明丝素蛋白水凝胶包载H₂S后达到缓释效果。CCK8结果显示H2S@SF具有较低细胞毒性。尼氏染色、FJB染色结果提示SCI后变性神经元数量明显增多,Na HS可抑制神经元变性,而H2S@SF效果更好;免疫荧光染色提示SCI后神经元数量降低,Na HS促进神经元存活,而H2S@SF效果更好;Westen blot及免疫荧光染色结果显示SCI后SYP表达降低,轴突功能被抑制,Na HS可以促进轴突功能恢复,而H2S@SF效果好。BBB评分及足迹实验结果显示Na HS促进了运动功能恢复,H2S@SF效果更好。第二部分:Westen blot结果显示焦亡相关蛋白（Caspase-1、GSDMD、ASC）呈现在1d时升高,2d时下降,3d时上升并达高峰的时序性特点;免疫荧光染色显示这些蛋白在SCI后神经元中阳性表达,表明SCI后神经元发生焦亡;Westen blot结果提示Na HS干预后焦亡相关蛋白表达均下调,H2S@SF下调更为显著;内源性H₂S合成酶相关蛋白CBS在SCI后表达升高,而CSE和3-MST表达无明显变化,Na HS干预后CBS表达下降,H2S@SF干预后下降更为显著;免疫荧光显示CBS在SCI后神经元中阳性表达并与GSDMD共定位;微型硫化物离子电极结果显示SCI后血液,脑脊液及脊髓组织中H₂S相对含量均下降,Na HS干预后升高;H2S@SF升高更为显著;焦亡抑制剂AC-YVAD-CMK干预后,Westen blot结果显示焦亡相关蛋白表达下调,CBS无明显变化,而CBS抑制剂AOAA提前干预后,H2S@SF对CBS及焦亡相关蛋白无调节作用。铁死亡相关蛋白检测的Westen blot结果显示SCI后COX2表达升高,GPX4和x CT表达下降,Na HS干预后COX2表达下调,GPX4和x CT表达上调;H2S@SF干预效果更为显著。第三部分:Westen blot结果显示SCI后PINK1、Parkin、P62表达升高,LC3Ⅱ/LC3Ⅰ表达下降;Na HS干预后PINK1、Parkin、P62表达下降,LC3Ⅱ/LC3Ⅰ表达升高,而H2S@SF与Na HS效果相似,无明显差异。免疫荧光显示PINK1在SCI后神经元中阳性表达并与GSDMD共定位;Rab7蛋白的Westen blot检测结果显示与焦亡相关蛋白呈相似时序性特点。免疫组化结果与Westen blot结果相一致;免疫荧光显示Rab7在SCI后神经元中阳性表达并与GSDMD共定位。焦亡抑制剂AC-YVAD-CMK干预后,Westen blot结果显示焦亡相关蛋白及Rab7表达均降低。研究结论1.我们成功制备了H₂S缓释的丝素蛋白水凝胶,且细胞毒性较低;外源性H₂S可以抑制SCI后神经元变性和死亡,促进轴突功能恢复,改善大鼠运动功能,而丝素蛋白水凝胶包载H₂S缓释后效果更好2.外源性H₂S抑制SCI后神经元焦亡和铁死亡,丝素蛋白水凝胶包载H₂S缓释后抑制效果更明显;且外源性H₂S可能通过CBS信号通路抑制SCI后神经元焦亡3.SCI后线粒体自噬被抑制,外源性H₂S可以促进PINK1/Parkin介导的线粒体自噬;在SCI病理生理发展过程中,线粒体自噬可能与细胞焦亡存在交互作用。更多还原

【Abstract】 Background and objectiveSpinal cord injury（SCI）is a destructive neuropathological disease,which can lead to severe motor,sensory and autonomic nerve dysfunction.The morbidity is high and brings a severe economic burden to the society.At present,effective therapeutic methods are still lacking.Complex pathomechanisms are the main reasons for the failure of treatment.Studies have shown that pyroptosis,ferroptosis,and mitophagy of SCI play an important role in the pathological process.Therefore,inhibiting cell death and promoting mitophagy may become a new strategy for the treatment of SCI.Hydrogen sulfide（H₂S）in the SCI has proven to have a neuroprotective effect,but the exact mechanism is unknown and needs to be explored.It is seldom reported whether H₂S affects pyroptosis,ferroptosis,and mitophagy.Furthermore,H₂S,a gas signal molecule,is highly volatile and cytotoxic due to local high concentrations caused by transient volatility,which also hinders its application prospects.Therefore,it is significant to find suitable carriers to load H₂S and make it release slowly,which can exert its effect better.Silk Fibroin hydrogel（SF-Gel）,a new natural protein material,has been widely used in the field of tissue engineering and regenerative medicine.It can significantly delay the half-life of some specific drugs when combined with some drugs.It may combine with HS-（the existing form of H₂S in vivo）and promote slowing the release rate down.In this study,we aimed to construct H₂S sustained-release SF-Gel and apply it to the rat-SCI model to observe its neuroprotective effect.In this process,we explored the regulatory effect of exogenous H₂S on pyroptosis,ferroptosis,and mitophagy in SCI,and understand the relationship between mitophagy and pyroptosis.This research aims to provide new ideas for the clinical treatment of SCI.MethodsPart Ⅰ:we utilized the physical dissolution of Na HS（H₂S donor）to mix with the SF-Gel to prepare the H₂S-releasing Silk Fibroin Hydrogel（H2S@SF）.To observe the characteristics of this H2S@SF,we utilized the emission scanning electron microscope to observe the situation of SF-Gel and H2S@SF.An Xplo RA laser Raman spectrometer was used to acquire Raman spectra of Na HS aqueous solution,SF hydrogel,and silk fibroin hydrogel with different concentrations（0.2um,2um,20um,200um）of H₂S.The Micro Sulfide Ion Electrode was used to detect H₂S release at different time points（1h,2h,4h,8h,24h,36h,48h and 72h）.The CCK8 test was used to detect the cytotoxicity of SF-Gel and H2S@SF.These rats were randomly divided into the Control group,SCI group,Na HS group,and H2S@SF group.The neuronal degeneration and survival were detected by using the Nissl staining,the FJB staining,and the immunofluorescence staining.The protein SYP was detected by the Western blot and immunofluorescence staining.The BBB score and footprinting test evaluated the recovery of motor function after injecting H2S@SF.Part Ⅱ:Rats were divided into the Control group and SCI groups（at different time points after injury,such as 1h,6h,12h,1d,2d,3d,7d）.The temporal changes of pyroptosis-related proteins（Caspase-1,GSDMD,ASC）were examined by Western blot.Immunofluorescence staining was used to analyze the cellular localization of these proteins.Then,rats were divided into Control,SCI,Na HS,and H2S@SF groups.Western blot was used to test the expression of pyroptosis-related proteins and endogenous H₂S synthetases.The immunofluorescence staining was used to analyze the cellular localization of protein CBS.The micro sulfide ion electrode was used to detect the relative levels of H₂S in spinal cord tissue,blood,and cerebrospinal fluid of rats in each group,and to evaluate the regulatory effect of exogenous H₂S and the effect of sustained release of H₂S@SF.To explore the regulatory mechanisms of H₂S in pyroptosis,pyroptosis-related proteins,and endogenous H₂S synthetase were tested after injecting the pyroptosis inhibitor（AC-YVAD-CMK）and the CBS inhibitor（AOAA）.Furthermore,ferroptosis-related proteins（GPX4,COX2,x CT）were tested among these groups.Immunofluorescence staining was used to detect the cellular localization of ferroptosis core protein GPX4,which was used to evaluate the regulatory effect of exogenous H₂S on ferroptosis and the sustained release of H2S@SF.Part Ⅲ:Proteins associated with mitophagy（LC3,PINK1,Parkin,P62）were detected among these groups（sham;SCI;Na HS,and H2S@SF）.Immunofluorescence staining was used to detect the cellular localization of protein PINK1,which was used to evaluate the regulatory effect of exogenous H₂S on mitophagy and the sustained release of H2S@SF.Then,protein Rab7 was detected among the Sham and SCI groups（at different time points after injury,such as 1h,6h,12h,1d,2d,3d,7d）using western blot and immunofluorescence staining.Furthermore,immunofluorescence staining was also used to detect the cellular localization.To explore the link between mitophagy and pyroptosis,the expression of Rab7was detected by western blot after injecting the pyroptosis inhibitor AC-YVAD-CMK.Results:Part Ⅰ:Electron microscopy shows H2S@SF has the characteristic of an open and interconnected porous morphology.Raman spectroscopy showed that the groups with SF-Gel showed a characteristic peak of silk fibroin（which was attributed to amideⅠ）at1662cm-1,indicating that the SF-Gel had an obviousβ-fold structure and the macromolecular conformation of silk fibroin in SF-Gel was stable.The characteristic peak at 2571 cm-1 was attributed to the-S-H-bond,and the characteristic peak became more obvious with the increase of Na HS carrying concentration in the gel,indicating that H₂S was effectively carried by the SF-Gel.The data of the micro-sulfide ion electrode showed that more than 90%of H₂S was released in Na HS solution within 2 hours and was released completely after 4 hours.However,H₂S in the H2S@SF was released at about 60%after 2hours and was released completely after 72 hours,indicating that H₂S could be sustainably released in the SF-Gel.The result of the CCK8 experiment showed the H2S@SF has the characteristic of low toxicity.Nissl staining and FJB staining showed that the number of degenerated neurons increased significantly after SCI.Na HS could inhibit neuronal degeneration,and H2S@SF hydrogel was more effective.Immunofluorescence staining showed that the number of neurons decreased after SCI,and Na HS promoted the survival of neurons,while H2S@SF was more effective.Western blot and immunofluorescence staining showed that the expression of SYP was decreased and the function of axons was inhibited after SCI.Na HS could promote the recovery of axon function,and H2S@SF had a better effect.BBB score and footprinting test showed that Na HS promoted the recovery of motor function,and H2S@SF had a better effect.Part Ⅱ:The results caused by the western blot showed that pyroptosis-related proteins（Caspase-1,GSDMD,ASC）increased on the first day and decreased on the second day.On the third day,these proteins increased and reached their peak.Immunofluorescence staining showed that these proteins were positively expressed in neurons,which indicated that neurons underwent pyroptosis after SCI.However,this situation was reversed after Na HS or H2S@SF intervention.Of which,the effect of H2S@SF was more obvious.The expression of endogenous H₂S synthetase-related protein CBS increased after SCI,while CSE and 3-MST had no significant changes.The expression of CBS after SCI was reversed after Na HS or H2S@SF intervention.Of which,the effect of H2S@SF was more obvious.Immunofluorescence staining showed that CBS was positively expressed in neurons and co-localized with GSDMD.The micro-sulfide ion electrode showed that H₂S in blood,cerebrospinal fluid,and spinal cord tissue decreased after SCI.However,this situation was reversed after Na HS or H2S@SF intervention.Of which,the effect of H₂S@SF was more obvious.After the intervention of pyroptosis inhibitor AC-YVAD-CMK,the Western blot showed that the expression of pyroptosis-related proteins was reduced,and there was no significant change in CBS.However,after early intervention with the CBS inhibitor AOAA,H2S@SF had no regulatory effect on CBS and pyroptosis-related proteins.Ferroptosis-related proteins were detected by Western blot.Protein COX2 was increased and protein GPX4 and x CT was decreased after SCI.However,the expression of these proteins（COX2,GPX4,and x CT）was reversed when giving Na HS or H2S@SF.Of which,the effect of H₂S@SF was more obvious.Part Ⅲ:Western blot showed that the expression of PINK1,Parkin,and P62 increased,and the ratio of LC3Ⅱ/LC3Ⅰdecreased after SCI.However,this result could be reversed when giving Na HS or H2S@SF intervention.Immunofluorescence showed that PINK1 was positively expressed in neurons and co-localized with GSDMD.Protein Rab7 increased on the first day and decreased on the second day after SCI.Moreover,it increased and reached its peak on the third day.This trend is consistent with pyroptotic proteins.This trend was consistent with that of pyroptotic proteins.Furthermore,Immunofluorescence staining showed the same result,and Rab7 was positively expressed in neurons and co-localized with GSDMD.The expression of Rab7 in SCI can be reversed when giving the pyroptosis inhibitor（AC-YVAD-CMK）.Conclusion:1.We successfully prepared H2S@SF with low cytotoxicity.Exogenous H₂S can inhibit neuronal degeneration and death,promote axon function recovery,and improve motor function after SCI.Compared with Na HS,H2S@SF is more effective.2.Exogenous H₂S inhibits neuronal pyroptosis and ferroptosis after SCI,of which H2S@SF showed an obvious inhibiting effect due to its sustained release capacity.Moreover,exogenous H₂S may inhibit pyroptosis through the CBS signaling pathway after SCI.3.Mitophagy was inhibited After SCI,and exogenous H₂S could promote PINK1/Parkin-mediated mitophagy.Furthermore,mitophagy may interact with pyroptosis in the pathological process of SCI.更多还原