Anti-inflammatory And Analgesic Targets And Mechanisms Of Cannabinoids

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.