【作者】 王雪；

【导师】 姜艳芳；

【作者基本信息】 吉林大学 ， 临床检验诊断学， 2024， 博士

【摘要】 背景:慢性疼痛是指持续或者反复发作超过3个月的疼痛。《中国疼痛医学发展报告(2020)》数据显示,我国慢性疼痛患者超过3亿人,且正以每年1000万至2000万的速度增长。慢性疼痛已成为继心脑血管疾病、肿瘤之后第三大健康问题,严重影响人们的健康和生活质量。神经病理性疼痛是难治的慢性疼痛。目前,临床上用于治疗神经病理性疼痛的药物主要包括阿片类药物(如吗啡),三环抗抑郁药,抗癫痫药。然而,这些药物临床疗效不佳且副作用较大。如何开发出镇痛效果好、副作用小、无成瘾耐受性的药物对神经病理性疼痛的治疗意义重大。现代医学研究证明,神经病理性疼痛的发生和演变与中枢神经系统(Central nervous system,CNS)的炎症密切相关。大麻二酚(Cannabidiol,CBD)及其类似物次大麻二酚(Cannabidivarin,CBDV)是两种植物大麻提取物,二者抗炎效果显著,特别是CBDV可能抗炎效果更佳。此外,CBD及其类似物CBDV具有良好的脂溶性,较易穿过血脑屏障,利于在中枢神经系统富集,并发挥药理作用。因此,CBD/CBDV可能适用于治疗神经病理性疼痛,然而其发挥镇痛作用的靶点及机制尚不清楚。在课题组的前期研究中,我们借助化学修饰的CBD小分子探针,通过点击化学和生物正交反应,发现FKBP5(FK506 binding protein 5)可能是CBD的作用靶点。因此,明确CBD的药物作用靶点及分子机制,可能有助于发掘新的具有临床应用价值的镇痛药物,还可以依据特异性靶点进而设计出与这些特异性靶点相互作用的CBD衍生物或类似物,从而改善疗效,增加生物利用度并减少副作用。此外,前期研究表明CBDV的抗炎效果优于CBD且机制不明。因此,我们还探讨了CBD类似物CBDV的药理作用机制及其逆转常见镇痛药物(吗啡)耐受的可能性。目的:1、研究CBD减轻神经炎症、发挥镇痛作用的靶点及机制;2、探索CBDV减轻神经炎症、改善吗啡镇痛作用、降低吗啡耐受的靶点及机制。方法:第一部分大麻二酚靶向FKBP5缓解大鼠神经病理性疼痛1、探究CBD与FKBP5的结合(1)Pull down实验:化学合成CBD光亲和探针CBD-P1并通过核磁鉴定。将CBD-P1与BV-2细胞裂解液共孵育,行点击化学反应后,Western Blot检测CBDP1与靶标蛋白FKBP5的结合情况。进一步将CBD-P1再分别与靶蛋白His-tagFKBP5或His-tag-FKBP5 Y113A共孵育,行点击化学反应后,Western Blot检测CBD-P1与靶标蛋白的结合情况。(2)荧光滴定实验:用不同浓度的CBD分别滴定靶蛋白FKBP5/FKBP5 Y113A,测量靶蛋白荧光强度的变化,验证小分子与靶标蛋白的结合情况。(3)热位移实验:将BV-2细胞裂解液与CBD共孵育,Western Blot检测不同温度梯度下靶标蛋白的含量变化。进一步将His-tag-FKBP5或His-tag-FKBP5Y113A再分别与CBD共孵育,Western Blot检测不同温度梯度下靶标蛋白的含量变化。以此验证小分子与靶标蛋白的结合情况。(4)分子对接实验:将靶标蛋白FKBP5与CBD进行构象对接,并预测二者结合的关键氨基酸残基。2、明确CBD在LPS诱导的BV-2细胞炎症模型中的作用首先构建细胞炎症模型。使用脂多糖(Lipopolysaccharide,LPS)处理BV-2细胞、HEK Blue h TLR4细胞、BV-2 dual细胞,以诱导细胞炎症模型,诱导的具体时间根据不同实验需求确定。(1)在已建立的细胞炎症模型下,首先通过免疫共沉淀和Western Blot技术验证CBD对FKBP5生物学功能及相关下游信号通路的影响。此外,我们还利用胚胎分泌碱性磷酸酶实验(Secreted embryonic alkaline phosphatase,SEAP assay)和双荧光素酶NF-κB报告实验(Dual-luciferase NF-κB reporter assay)分别检测CBD对HEK Blue h TLR4细胞和BV-2 NF-κB荧光素酶报告细胞的NF-κB活性影响。(2)在已建立的细胞炎症模型下,我们还使用比色法检测CBD对下游一氧化氮(Nitric oxide,NO)水平的影响,同时使用q RT-PCR检测炎症相关的基因(TNF-α、IL-1β、IL-6)m RNA表达水平。此外,我们构建了突变稳转细胞系,细胞系中内源性FKBP5被敲低,同时表达与内源性FKBP5的水平相当的FKBP5 Y113A,并通过Western Blot鉴定。在此细胞系的炎症模型下,我们检测了CBD对下游炎症因子的影响。3、研究CBD对神经病理性疼痛的影响及机制暴露大鼠左侧坐骨神经,在坐骨神经周围系4个松结,对坐骨神经造成慢性压迫损伤(Chronic constriction injury of the sciatic nerve,CCI)。CCI模型构建14天后,测定大鼠手术侧足机械痛阈值,<8 g即为造模成功,可用于之后的实验。通过行为药理学方法观测CBD对CCI模型大鼠机械刺激阈值的影响,并使用免疫荧光化学的方法观察脊髓背角相关细胞和分子在慢性疼痛过程中的变化。第二部分次大麻二酚靶向TLR4辅助蛋白MD2改善吗啡镇痛作用1、验证CBDV与MD2的结合(1)荧光滴定实验:用不同浓度的CBDV滴定靶蛋白MD2,测量靶蛋白荧光强度的变化,验证小分子与靶标蛋白的结合情况。(2)细胞热位移实验:将BV-2细胞裂解液与CBDV共孵育,Western Blot检测不同温度梯度下靶标蛋白MD2的含量变化,以此验证小分子与靶标蛋白的结合情况。(3)表面等离子共振分析:将MD2蛋白与SR7000传感器芯片偶联。利用光在不同介质中产生消逝波后与等离子波产生共振的原理,测试小分子CBDV与MD2蛋白的结合力。(4)计算模拟与分子对接实验:将靶标蛋白MD2与CBDV进行构象对接,并预测二者结合的关键氨基酸残基。同时,模拟二者相互结合后MD2蛋白的构象变化,计算二者结合后稳态的结合能。2、探索CBDV在LPS诱导的BV-2细胞炎症模型中的作用首先构建细胞炎症模型,具体方法与第一部分相同。(1)在已建立的细胞炎症模型下,我们首先通过免疫共沉淀和Western Blot验证CBDV对MD2下游信号通路相关蛋白含量的影响。此外,我们还利用SEAP assay和Dual-luciferase assay分别检测CBDV对HEK Blue h TLR4细胞和BV-2NF-κB荧光素酶报告细胞的NF-κB活性影响。(2)在已建立的细胞炎症模型下,我们还使用比色法检测CBDV对下游一氧化氮(Nitric oxide,NO)的水平影响,同时使用q RT-PCR检测炎症相关的基因(TNF-α、IL-1β、IL-6)的m RNA含量。3、探究CBDV对吗啡耐受的影响及机制(1)将小鼠置于55℃热板仪上,并密切观察小鼠热应激后的反应同时迅速记录反应时间,构建以上小鼠热板模型并验证模型构建成功后,可用于后续实验。(2)用27号针将20μL 2%福尔马林注射到小鼠左后爪中,构建小鼠福尔马林疼痛模型,分别记录小鼠在急性期和慢性期舔咬爪子的秒数,验证模型构建成功后,可用于后续实验。(3)连续7天,每日一次腹腔注射吗啡1 mg/kg,构建小鼠吗啡耐受模型。在注射吗啡后的第1天和第7天进行福尔马林疼痛实验,验证模型成功,可用于后续实验。(4)成功构建以上三种动物模型验证后,通过行为药理学方法观测CBDV对热痛、化学痛及吗啡耐受的影响。行为实验结束后,免疫荧光染色检测m PFC、VTA、NAc脑区胶质细胞的变化。同时,使用q PCR检测相关脑区炎症因子的表达水平。结果:第一部分大麻二酚靶向FKBP5缓解大鼠神经病理性疼痛1、CBD与FKBP5直接结合(1)通过体外Pull down实验、荧光滴定实验、热位移实验证明了CBD与FKBP5直接结合(2)通过分子对接实验证明FKBP5与CBD直接结合,二者的结合位点为FKBP5Y113。2、CBD减轻LPS诱导的BV-2细胞炎症(1)CBD抑制LPS诱导的IKK(IκB kinases)复合物(IKKα、IKKβ和IKKγ)募集及NF-κB的激活,同时抑制LPS诱导的下游IκB和p65的磷酸化。(2)CBD抑制LPS诱导的促炎因子(NO、IL-1β、IL-6和TNF-α)表达,其发挥抗炎作用的靶点是蛋白FKBP5的Y113位点。3、CBD可以减轻神经病理性疼痛(1)CBD可以减轻神经病理性疼痛:行为学实验表明,CBD以剂量和时间依赖的方式降低了CCI诱导的机械超敏反应。(2)CBD抑制小胶质细胞中FKBP5的表达:免疫荧光结果显示,CBD抑制了CCI诱导的脊髓背角活化小胶质细胞中FKBP5的过表达,这可能是CBD减轻大鼠的神经病理性疼痛的机制。第二部分次大麻二酚靶向TLR4辅助蛋白MD2改善吗啡镇痛作用1、CBDV与MD2直接结合(1)通过体外荧光滴定实验、细胞热位移实验、表明等离子共振分析实验证明了CBD与FKBP5直接结合(2)通过计算模拟与分子对接实验证明CBDV与MD2直接结合,稳态结合能为5.5?。二者结合的关键位点分别为Ile32、Cys51、Ile52、Val61、Phe76、Leu78、Phe119、Cys133、Ala135、Ile153。2、CBDV减轻LPS诱导的BV-2细胞炎症(1)CBDV抑制TLR4/MD2/My D88复合物形成和NF-κB活性,同时抑制下游信号通路转导。(2)CBDV抑制下游炎症因子(NO、IL-1β、IL-6和TNF-α)产生。3、CBDV改善吗啡介导的镇痛作用(1)在小鼠热板模型、小鼠福尔马林疼痛模型和吗啡耐受模型中证明,CBDV增加和延长吗啡镇痛、降低吗啡耐受。(2)CBDV可以抑制吗啡耐受模型中NAc脑区小胶质细胞和星形胶质细胞的活化,同时抑制炎症因子的表达。结论:1、细胞实验和动物实验结果证明,大麻二酚(CBD)靶向FKBP5缓解大鼠神经病理性疼痛。FKBP5第113位酪氨酸残基是FKBP5与CBD结合的关键位点。2、本研究发现了CBD的类似物次大麻二酚(CBDV)是TLR4的拮抗剂。细胞实验和动物实验结果证明,CBDV在低剂量(0.5μM)下即可直接靶向TLR4辅助蛋白MD2,改善小鼠吗啡耐受。更多还原

【Abstract】 Background:Chronic pain is pain that persists or recurs for more than 3 months.According to the "China Pain Medicine Development Report(2020)",there are more than 300 million people with chronic pain in China,and it is growing at a rate of 10 million to 20 million per year.Chronic pain has become the third major health problem after cardiovascular and cerebrovascular diseases and tumors,which seriously affects people’s health and quality of life.Neuropathic pain is a common chronic pain.At present,the drugs used in the clinical treatment of neuropathic pain mainly include opioids(such as morphine),tricyclic antidepressants,and antiepileptic drugs.However,these drugs have poor clinical efficacy and side effects.How to develop drugs with good analgesic effect,few side effects and no addiction tolerance is of great significance for the treatment of neuropathic pain.It is well known that the occurrence and evolution of neuropathic pain are closely related to the inflammation of the Central nervous system(CNS).Cannabidiol(CBD)and its analogue Cannabidivarin(CBDV)are two plant cannabis extracts,both of which have significant anti-inflammatory effects,especially CBDV may have a better antiinflammatory effect.In addition,CBD and its analogue CBDV have good lipid solubility and can easily cross the blood-brain barrier,which is conducive to enrichment in the central nervous system and exert pharmacological effects.Therefore,CBD/CBDV may be suitable for the treatment of neuropathic pain.However,the analgesic targets and mechanisms of CBD/CBDV in the treatment of neuropathic pain are still unclear.In order to find the pharmacological target of CBD,our previous research group used chemically modified CBD small molecule probe,and found that FKBP5(FK506X binding protein 5)may be the target of CBD through click reaction and biological orthogonal reaction.Therefore,we validated the results at the cellular and animal levels and further explored the downstream mechanisms.In addition,preliminary studies have shown that CBDV has better anti-inflammatory effects than CBD,but the results of existing studies are limited and the mechanism is unclear.Therefore,we also explored the pharmacological mechanism of CBDV and the possibility of reversing the tolerance of common analgesics(morphine).Objective:1.Investigate the targets and mechanisms through which CBD alleviates neuroinflammation and exerts its analgesic effects.2.Explore the targets and mechanisms through which CBDV reduces neuroinflammation,improves opioid analgesia,and reduces opioid tolerance.Methods:Part 1.Cannabidiol(CBD)targeting FKBP5 for alleviating rat neuropathic pain1.Investigating the binding of CBD to FKBP5(1)Pull-Down assays: Incubating CBD-P1 with BV-2 cell lysates,performing click chemistry reactions,and detecting the binding of CBD-P1 to the target protein FKBP5 via Western Blot.Similarly,incubating CBD-P1 with His-FKBP5 and His-FKBP5Y113 A,performing click chemistry reactions,and detecting the binding of CBD-P1 to the target protein via Western Blot..(2)Fluorescence titrations: Titrating FKBP5/FKBP5 Y113 A with varying concentrations of CBD and measuring changes in fluorescence intensity of the target protein to validate the binding of the small molecule to the target protein.(3)Cellular thermal shift assays: Incubating BV-2 cell lysates with CBD and performing Western Blot to assess changes in the target protein’s content at different temperature gradients,confirming the binding of the small molecule to the target protein.Similar experiments can be conducted with the target proteins His-tag-FKBP5 or His-tag-FKBP5 Y113 A incubated with CBD,performing Western Blot to assess changes in the target protein’s content at different temperature gradients,confirming the binding of the small molecule to the target protein.(4)Molecular docking: Performing conformational docking of the target protein FKBP5 with CBD and predicting key amino acid residues involved in their binding.2.Elucidating the effects of CBD in the LPS-induced inflammatory model of BV-2 Cells(1)Establishment of cell inflammatory models: BV-2 cells,HEK Blue h TLR4 cells,and BV-2 dual cells are subjected to lipopolysaccharide(LPS)treatment to induce cell inflammatory models.The specific induction duration is determined based on the experimental requirements.(2)In the established cell inflammatory models,the impact of CBD on the biological function of FKBP5 and related downstream signaling pathways is initially validated using immunoprecipitation and Western Blot techniques.Additionally,the influence of CBD on NF-κB activity is assessed separately using Secreted Embryonic Alkaline Phosphatase(SEAP)assays for HEK Blue h TLR4 cells and Dual-Luciferase NF-κB reporter assays for BV-2 NF-κB luciferase reporter cells.(3)Within the established cell inflammatory models,CBD’s effects on downstream nitric oxide(NO)levels are evaluated using a colorimetric assay.Furthermore,the m RNA expression levels of inflammation-related genes(TNF-α,IL-1β,IL-6)are assessed using quantitative real-time polymerase chain reaction(q RT-PCR).Additionally,a stable mutant cell line is generated with knocked-down endogenous FKBP5 expression,concomitantly expressing FKBP5 Y113 A at levels comparable to endogenous FKBP5.In this cell line,we also examine the levels of downstream XII inflammatory factors.3.Investigating the effects and mechanisms of CBD on neuropathic pain Rats are subjected to exposure of the left sciatic nerve,and four loosely tied ligatures are placed around the sciatic nerve to induce chronic constriction injury(CCI).Fourteen days after the establishment of the CCI model,the mechanical pain threshold of the rat’s operated hind paw is measured,with a threshold of <8 g indicating a successful model induction,which is subsequently used for further experiments.The impact of CBD on the mechanical stimulus threshold in CCI model rats is observed using behavioral pharmacological methods.Additionally,changes in spinal dorsal horn-related cells and molecules during chronic pain processes are examined through immunofluorescence chemistry.Part Two: CBDV targeting TLR4 co-receptor MD2 to improve morphine Analgesia1.Validating the binding of CBDV to MD2(1)Fluorescence titration assay: Titration of MD2 with CBDV at different concentrations to measure changes in MD2 fluorescence intensity,confirming the binding of the small molecule to the target protein.(2)Cell thermal shift assay: Incubation of BV-2 cell lysate with CBDV to assess changes in the MD2 protein’s content at different temperature gradients using Western Blot,verifying the interaction between the small molecule and the target protein.(3)Surface plasmon resonance analysis: MD2 protein was coupled to SR7000 sensor chip.The binding force between small molecules and proteins is tested by using the principle that light generates evanescent waves in different media and then resonates with plasma waves.(4)Computational modeling and molecular docking: Performing structural docking of MD2 with CBDV and predicting key amino acid residues involved in their binding.Additionally,simulating the conformational changes in MD2 protein after binding with CBDV and calculating the binding energy to reach a stable state.2.Understanding of the effects of CBDV in LPS-induced BV-2 cell inflammatory model(1)Construction of the cell inflammatory model: Same as the Part one.(2)In the established cell inflammatory model,initial validation of CBDV’s impact on downstream signaling pathway-related proteins from MD2 is conducted through coimmunoprecipitation and Western Blot analysis.Furthermore,CBDV’s effect on NF-κB activity is assessed in HEK Blue h TLR4 cells and BV-2 NF-κB luciferase reporter cells using SEAP assay and Dual-luciferase assay,respectively.(3)In the established cell inflammatory model,the influence of CBDV on downstream nitric oxide(NO)levels is measured using a colorimetric assay,and the m RNA expression levels of inflammation-related genes(TNF-α,IL-1β,IL-6)are evaluated through q RT-PCR.3.Clarifying the impact and mechanisms of CBDV on morphine tolerance(1)Mice are placed on a 55°C hot plate,and their reactions to heat stress are closely observed,with the reaction time promptly recorded.The thermal pain model in mice is constructed and confirmed successful for subsequent experiments.(2)A 20 μL injection of 2% formalin is administered into the left hind paw of mice using a 27-gauge needle to create a formalin pain model.The duration of paw licking and biting is recorded in both the acute and chronic phases to validate the successful construction of the model for subsequent experiments.(3)Mice are subjected to daily intraperitoneal injections of morphine at 1 mg/kg for seven consecutive days to establish a morphine tolerance model.To verify the morphine XIV tolerance,a formalin pain test is conducted on the 1st and 7th days following morphine injections.Upon successful model validation,it is ready for subsequent experiments.(4)After successfully constructing the three animal models and confirming their validity,the effects of CBDV on thermal pain,chemical pain,and morphine tolerance are observed through behavioral pharmacology methods.Following behavioral experiments,immunofluorescence staining is performed to examine changes in glial cells in relevant brain regions.Additionally,q PCR is used to assess the expression levels of inflammatory factors in the brain regions.Results:Part One: CBD targeting FKBP5 alleviates neuropathic pain in rats1.Biophysical interaction between CBD and FKBP52.CBD reduced LPS-induced inflammation in BV-2 cells(1)CBD inhibits the recruitment of the LPS-induced IKK complex(IKKα,IKKβ,and IKKγ).CBD effectively inhibits the activation of downstream pathways of the LPSinduced IKK complex.CBD inhibits the activation of LPS-induced NF-κB.(2)CBD inhibits LPS-induced proinflammatory cytokines(NO,IL-1β,IL-6,and TNF-α).Y113 residue of FKBP5 is the key site for the interaction with CBD.3.CBD alleviates neuropathic pain(1)CBD alleviates neuropathic pain: Behavioral experiments reveal that CBD reduces the mechanical hypersensitivity induced by CCI in a dose and time-dependent manner.(2)CBD inhibits the expression of FKBP5 in activated microglia: Immunofluorescence results demonstrate that CBD suppresses the overexpression of FKBP5 in activated microglia induced by CCI,which is a potential mechanism for CBD’s alleviation of neuropathic pain in rats.Part Two: CBDV targeting TLR4 co-receptor MD2 improves morphine analgesia1.Biophysical interaction between CBDV and MD22.CBDV reduced LPS-induced inflammation in BV-2 cells(1)CBDV inhibits the formation of TLR4/MD2/My D88 complex.CBDV inhibits the TLR4 signaling pathways NF-κB and MAPK.CBDV effectively inhibits the activation of NF-κB induced by LPS.(2)CBDV inhibits the production of proinflammatory cytokines downstream of the TLR4 signaling pathway: CBDV inhibits the elevation of NO,IL-1β,IL-6,and TNF-αinduced by LPS.4.CBDV enhances morphine analgesia:(1)In mouse hot plate,formalin,and morphine tolerance models,CBDV increases and prolongs morphine analgesia while reducing morphine tolerance.(2)CBDV suppresses the activation of microglia and astrocytes in the morphine tolerance model and inhibits the expression of inflammatory factors.Conclusion:1.In vitro and cellular experiments demonstrate that CBD inhibits inflammation by targeting FKBP5,which inhibits the assembly of the IKK complex and subsequently suppresses downstream inflammatory pathways.Tyrosine 113 of FKBP5 is identified as the critical binding site for CBD.Animal experiments further confirm these findings and show that CBD can inhibit the overexpression of FKBP5 in the spinal cord dorsal horn microglia of CCI rats,thus alleviating neuropathic pain.2.This study further reveals that CBDV,a CBD analogue,acts as an antagonist of TLR4.In vitro experiments confirm that CBDV can inhibit TLR4/MD2 signaling pathway transduction at low concentrations(0.5 μM)by directly targeting TLR4 coreceptor MD2.Animal experiments further demonstrate that CBDV specifically inhibits morphine-induced neuroinflammation,enhances morphine analgesia,and mitigates morphine tolerance.3.The precise mechanisms and targets of CBD and CBDV have been elucidated,contributing to a better understanding of their pharmacological properties and potential therapeutic applications.更多还原