Bone cancer pain is a chronic intractable cancer pain, which is often treated with opioids such as morphine in clinic, but long-term use of morphine is prone to produce tolerance, that is, morphine tolerance, which leads to the gradual weakening or even disappearance of the analgesic effect of morphine, and it needs to increase the dose of morphine to obtain the same analgesic effect[1],μOpioid receptor（μ opioidreceptor，MOPR）It is the most important factor for opioids to exert analgesic effect, whenMOPRWhen bound to opioid agonists, its molecular conformation changes, which is consistent withGProtein binding formationGprotein-Opioid receptor complex, which reduces intracellular cyclic adenosine monophosphate（cyclic adenosine monophosphate，cAMP）Equal signal concentration, prevent the conduction of pain impulse and exert analgesic effect[2]However, long-term use of opioids can causeMOPRProtein phosphorylation, leading toMOPRDesensitization, which in turn causes morphine tolerance. Therefore, exploring effective drugs and mechanisms of action to improve morphine tolerance in patients is an urgent problem in the clinical treatment of bone cancer pain, which is of great significance to improve the prognosis of patients with bone cancer pain.
β-Elemene is extracted from the natural Chinese herbal medicine curcuma wenyujinCurcuma wenyujin Y. H. Chen et. LingTerpenes extracted from Chinese herbal medicine are the main active ingredients of this kind of traditional Chinese Medicine[3-4]. the study found that beta-As a non cytotoxic antitumor drug, elemene has a "dual effect" of selectively inhibiting tumor cell proliferation and improving immune function[5]In addition, according to Chinese Pharmacopoeia, turmeric extract can "treat amenorrhea and dysmenorrhea, chest and abdomen pain, tingling, fever and dizziness, epilepsy and madness, jaundice and hematuria"[6]According to the Xinhua compendium of Materia Medica, the extract of Curcuma "can cure the symptoms such as the accumulation of symptoms and pains, blood stagnation, abdominal distension and pain, food accumulation and pain, and abdominal pain due to amenorrhea"[7]It can be seen that in addition to the anti-tumor effect, the extract of Curcuma also has the "analgesic" effect. Another study found that,β-Elemene may act by inhibitingN-methyl-D-Aspartate receptor subunit2B（N-methyl-D-asparate receptor subunit 2B，NR2B）Expression, regulationMOPR、cAMPAnd other factors to exert analgesic effect[8]However, at presentBeta elemeneThe efficacy and specific mechanism of morphine tolerance improvement in patients with bone cancer pain remain to be studied. In addition, ifenprodil is a kind of drug that can be used to treat the patients with chronic hepatitis B.the drug can be used to treat the patients with chronic hepatitis B.the drug can be used to treat the patients with chronic hepatitis B.the drug can be used to treatN-methyl-D-Aspartic acid（N-methyl-D-asparate，NMDA）Antagonists of receptors, onNR2B. Therefore, ifenprodil was used as a positive control drug in this studyBeta elemeneObjective to investigate the effect and mechanism of morphine tolerance in rats with bone cancer pain, and to provide a scientific basis for the clinical treatment of bone cancer pain.
1 material
1.1 animal
SPFGrade maleWistarRat75Only,6～8Weeks old, body mass（250±15）g, purchased from Shanghai slaker laboratory animal Co., Ltd., animal Certificate No[SCXK(Shanghai)0036875]The animals were kept inSPFLevel animal room,12 h/12 hAlternating light and dark, temperature（20±2）℃, humidity40%～60%The animal experiments were approved by the ethics committee of Zhejiang Cancer Hospital (approval no2020-110-003）, following the Chinese animal care and institutional ethics guidelines.
1.2 cell line
Rat breast cancerWalker 256Cells were purchased fromATCCCell bank, human neuroblastomaSH-SY5YCells were purchased fromICLC-IST.
1.3 Drugs and reagents
Morphine hydrochloride injection (batch No040203, Guoyao Zhun ZiH21022436，10 mg/mL）It was purchased from Shenyang first pharmaceutical Co., Ltd. of Northeast Pharmaceutical Group;β-Elemene (batch No0408251, Guoyao Zhun ZiH20110114）Purchased from Dalian Shiyao group Yuanda Pharmaceutical Co., Ltd; Ifenprodil (batch No12892）Purchased from USASigma-AldrichCompany; （1.5 mL/Piece, batch No200801255, Guoyao Zhun ZiS10870001）Purchased from Beijing Institute of Biological Products Co., Ltd; Protein extraction kit (batch NoBC3710）Purchased from Beijing solabo Biotechnology Co., Ltd;BCAProtein concentration assay kit (batch NoAB102536）Purchased from aibokang Shanghai Trading Co., Ltd;TrizolReagent (batch No15596026）Reverse transcription Kit (batch No4374966）Purchased from USAThermo Fisher ScientificCompany;CCK-8Kit (batch Noab228554）、MOPRPrimary antibody (batch Noab5392）、NR2BPrimary antibody (batch Noab254356）、cAMPPrimary antibody (batch Noab76238）、β-actinPrimary antibody (batch Noab8226）、HRPLabeled Goat anti rabbitIgGAntibody (batch Noab6721）、HRPLabeled Goat anti mouseIgGAntibody (batch Noab205719）Purchased from UKAbcamCompany.
1.4 instrument
371typeCO2IncubatorST16RType a high-speed refrigerated centrifugeMultiskan FcType microplate reader (USAThermo Fisher Scientific;DYCZ-20EType I electrophoresis equipment (Beijing Liuyi Biotechnology Co., Ltd.);LightCycler 96typeqRT-PCRInstrument (Roche Group); ITC Life Science）。
2 method
2.1 Animal grouping、Modeling and Administration
According to the random number table methodWistarRats were divided into control group, morphine group andβ-Elemene low and high dose（0.7、2.8 mg/kg）Group and ifenprodil（5 mg/kg）Group, each group15Only. The control group was injected with normal saline into the tibia, and the rats in the other groups were treated with bone cancer pain-Chronic morphine tolerance model[9]: firstly, the rat bone cancer pain model was establishedip 7%Sodium pentobarbital（3 mL/kg）After anesthesia, the abdomen was downward and the left hind limb was used first10 mLThe syringe was punctured and punched, and then changed10 μLA micropipette was introduced into the tibial bone marrow cavity and slowly injected3 μL Walker 256Breast cancer cell suspension（3×103～3×104After the injection, the needle hole was sealed with bone wax, the wound was cleaned, and the muscle skin was sutured; After that, the chronic morphine tolerance model was established, and the rats were inoculated with cancer cells10 dWhen, waist3～4The intrathecal tube was inserted into the puncture sheath of intervertebral space and morphine was injected（20 µL/kg），1second/d, continuous10 d, rats were injected with morphine10 d. Then the control group and morphine groupip 200 μLDimethyl sulfoxide（dimethyl sulfoxide，DMSO），Each dosing groupipSoluble in200μL DMSOCorresponding drugs,1second/d，continuity10 dAfter the behavioral measurement of rats in each group,ip2%After deep anesthesia with sodium pentobarbital, the rats were decapitated quickly and sacrificed, and the waist was removed on iceL4、L5A tissue specimen from the dorsal horn of the spinal cord.
2.2 Behavioral assessment of pain
After successful rat modeling1～10 dAccording to the following indicators, the pain behavior changes of rats were observed and recorded.
2.2.1 Mechanical withdrawal threshold detection The rats were placed in a glass plate chamber and placed on a metal screen to allow the rats to adapt to the environment10 minAfter that, the fine fibers of the stimulator were contacted with the middle part of the left plantar of the rat in a quiet and awake state, and the5 sCan be added up to50 gStrength, the paw withdrawal reaction of rats was observed and the mechanical withdrawal threshold was recorded.
2.2.2 Detection of heat withdrawal latency The rats were placed in a glass plate chamber, and the focal length of the light source of the thermal pain stimulator was adjusted to irradiate the palm of the hind limb of the animal in a quiet and awake state. The time from irradiation to paw withdrawal escape, namely the heat withdrawal latency, was recorded. Each rat was measured5Times, interval time5 min, upper limit20 s, to avoid causing tissue damage.
2.3 qRT-PCRDetection of tumor necrosis factor in rat spinal dorsal horn（tumor necrosis factor-α，TNF-α）、Interleukin-1β（interleukin-1β，IL-1β）、IL-6、NR2B、cAMPandMOPROfmRNAexpress
Rats in each group were takenSpinal dorsal horn tissue, fully cut the tissue, and extract the total according to the kit instructionsRNAAnd synthesizedcDNA, performedqRT-PCR. Reaction conditions:95 ℃ pre denaturation3 min, and then95 ℃ pre denaturation10 s，60 ℃ annealing30 s，70 ° C extension30 s；40Cycles. Primer sequence:GAPDHUpstream primer5’-GCAAGTTCAACGGCACAG-3’, downstream primers5’-CGCCAGTAGACTCCACGAC-3’；NR2BUpstream primer5’-TCCACA ATTA CICCTCGACG-3’, downstream primers5’-TCCGATTCTTCTICTGAGCC-3’；MOPRUpstream primer5’-TGCTCCTGGCTCAACTTGTCC-3’, downstream primers5’-GCGTGCTAGTGGCTAAGGCATCTG-3’；cAMPUpstream primer5’-AGGTCCTCAGCTACAAGGAAG-3’，Downstream primers5’-TCTTGAAGTCACAATCCTCTGGT-3’；TNF-αUpstream primer5’-CACCGGCAAGGATTCCAA-3’，Downstream primers5’-CACTCAGGCATCGACATTCG-3’；IL-1βUpstream primer5’-AGCCTTTGTCCTCTGCCAAGT-3’，Downstream primers5’-CCAGAATGTGCCACGGTTTT-3’；IL-6Upstream primer5’-TGTTCTCAGGGAGATCTTGGAAAT-3’，Downstream primers5’-CATCGCTGTTCATACAATCAGAATT-3’。
2.4 Western blottingDetection of spinal dorsal horn tissue in ratsNR2B、cAMPandMOPRProtein expression
Protein was extracted from rat spinal dorsal horn using a protein extraction kit according toBCAThe protein concentration was determined by protein quantification kit, and the protein samples were treated with sodium dodecyl sulfate-Polyacrylamide gel electrophoresis, go toPVDFMembrane,to5%Sealed in skimmed milk1 h, respectivelyNR2B（1∶1000）、cAMP（1∶50 000）、MOPR（1∶20 000）Andβ-actin（1∶1000）Antibodies,4 Incubate overnight at ° C; Secondary antibodies were added（1∶2000）, incubation1 h, usingECLChemiluminescence development, usingImage JThe software analyzes the band gray value.
2.5 In vitro experiments
2.5.1 cell culture SH-SY5YCells were seeded in medium containing100 IU/mLPenicillin100 μg/mLStreptomycin10%Fetal bovine serumDMEMIn high glucose medium, in37 ℃、5% CO2The cells were cultured in a cell incubator to logarithmic growth phase.
2.5.2 CCK-8Cell viability was measured by SH-SY5YCells in3×104/mLInoculated to96In the orifice plate, each hole100 µL, adding different mass concentrations（5、10、20、25 µg/mL）Ofβ-Elemene, the control group was added with drug free medium, and cultured48 hAfter that, each well was added10 μL CCK-8Solution, incubation4 h, determined by microplate reader450 nmAbsorbance at（A）.
2.5.3 IntracellularcAMPContent detection SH-SY5YCells in3×104/mLInoculated to96In the orifice plate, each hole100 µL, adding different mass concentrations（5、10、20、25 µg/mL）Ofβ-Elemene, the control group was added with drug free medium, and cultured48 hAfter that, the assay was performed according to the kit instructionscAMPContent.
2.5.4 Construction and administration of morphine tolerance cell model The control group was treated with conventional culture, and the control group was treated with10 µmol/LMorphine acting cells48 hTo construct a morphine tolerant cell model[10]At the same time, each dosing group was addedβ-（5 µg/mL）And ifenprodil（10 µmol/L）, role48 h。
2.5.5 qRT-PCRDetection of cellsTNF-α、IL-1β、IL-6、NR2B、cAMPandMOPROfmRNAexpress Press“2.5.4”Cells were treated according to the method under“2.3”Total withdrawal by method underRNAAnd synthesizedcDNA, performedqRT-PCRAnalysis.
2.5.6 Western blottingDetection of cellsNR2B、cAMPandMOPRProtein expression Press“2.5.4”Cells were treated according to the method under“2.4”Protein extraction and detection underNR2B、cAMPandMOPRProtein expression。
2.6 statistical analysis
applyGraphPad Prism 8.0Carry out statistical analysis and measure data withIndicated that multiple comparisons between groups were performed using one-way ANOVA（One-way ANOVA）, pairwise comparison between groupsLSDInspection.
3 result
3.1 Pain behavior assessment of rats in each group
As shown in Fig1As shown in, in the construction of rat bone cancer pain-Before chronic morphine tolerance model（0 d）There were no significant differences in mechanical withdrawal threshold and heat withdrawal latency among the groups; Administration section1Days（1 d）Compared with the control group, the mechanical withdrawal threshold and heat withdrawal latency of the other groups were significantly increased（P＜0.001）; The mechanical withdrawal threshold, heatThe withdrawal latency gradually decreased,β-The mechanical withdrawal threshold and heat withdrawal latency of elemene low-dose, high-dose and ifenprodil groups were significantly higher than those of morphine group（P＜0.05、0.01、0.001）; To page10Days（10 d）There was no significant difference in mechanical withdrawal threshold and heat withdrawal latency between morphine group and control group,β-The mechanical withdrawal threshold and heat withdrawal latency of elemene low-dose, high-dose and ifenprodil groups were significantly higher than those of morphine group（P＜0.01、0.001），β-The mechanical withdrawal threshold and heat withdrawal latency in the high-dose elemene group were higher thanβ-Elemene low dose group, descriptionβ-Both elemene and ifenprodil can effectively improve morphine tolerance in rats, andβ-When the dosage of elemene increases, the effect is better.
3.2 Rats in each groupSpinal dorsal horn tissueTNF-α、IL-1β、IL-6、NR2B、cAMPandMOPROfmRNAAnd proteinsurfacereach
As shown in Fig2-A、BAs shown in, compared with the control group, the spinal cord tissue of rats in the morphine groupTNF-α、IL-1β、IL-6、NR2BandcAMP mRNAThe expression level was significantly increased（P＜0.001），MOPR mRNAThe expression level was significantly reduced（P＜0.001）; Compared with the morphine group,β-Spinal cord tissue of elemene low and high dose groups and ifenprodil groupTNF-α、IL-1β、IL-6、NR2BandcAMP mRNAThe expression level was significantly reduced（P＜0.01、0.001），MOPR mRNALevels were significantly elevated（P＜0.001）. As shown in Fig2-CAs shown, compared with the control group, the spinal cord tissue of morphine groupNR2BandcAMPThe protein expression level was significantly increased（P＜0.001），MOPRThe protein expression level was significantly reduced（P＜0.001）; Compared with the morphine group,β-Spinal cord tissue of elemene low and high dose groups and ifenprodil groupNR2BandcAMPThe protein expression level was significantly reduced（P＜0.01、0.001），MOPRThe protein expression level was significantly increased（P＜0.001）。
3.3 Different concentrations ofβ-Elemene pairSH-SY5YCell viability andcAMPInfluence of content
Using different mass concentrations ofβ-Elemene treatment of normal culturedSH-SY5YCells and usedCCK-8Method detectionSH-SY5YCell viability, and the results are shown in Fig3-A，β-Elemene significantly inhibitedSH-SY5YCellular activity（P＜0.001）And showed dose and time correlation. Therefore, to5 µg/mL β-Elemene actionSH-SY5Ycell48 hFor the best experimental conditions. adoptcAMPAssay kitSH-SY5YcellcAMPThe results are shown in Fig3-B，β-Elemene pairSH-SY5YIntracellularcAMPThe content has no obvious effect.
3.4 β-Morphine tolerant cell model of elemenecAMPcontentandTNF-α、IL-1β、IL-6、NR2B、cAMPandMOPROfmRNAAnd proteinsurfaceInfluence of Da
The control group was given conventional culture, the morphine groupβ-Elemene group and ifenprodil group were treated with10 µmol/LMorphine actionSH-SY5Ycell48 h, to construct a morphine tolerant cell model, and thenβ-Elemene group and ifenprodil group were givenβ-Elemene, ifenprodil treatment, usingcAMPThe detection kit was used to detect the cells in each groupcAMPAssay, as shown in Fig4-AShown, compared with the control group, the morphine groupcAMPLevels were significantly elevated（P＜0.001; compared with the morphine group,β-Elemene, ifenprodil groupcAMPLevels were significantly reduced（P＜0.001）. As shown in Fig4-B、CShown, compared with the control group, the morphine groupTNF-α、IL-1β、IL-6、NR2BandcAMP mRNAThe expression level was significantly increased（P＜0.001），MOPR mRNAThe expression level was significantly reduced（P＜0.001）; Compared with the morphine group,β-Elemene group, ifenprodil groupTNF-α、IL-1β、IL-6、NR2BandcAMP mRNAThe expression level was significantly reduced（P＜0.001），MOPR mRNAThe expression level was significantly increased（P＜0.001）. As shown in Fig4-D、EShown, compared to theMorphine groupNR2BandcAMPThe protein expression level was significantly increasedHigh（P＜0.001），MOPRThe protein expression level was significantly reduced（P＜0.001）; Compared with the morphine group,β-Elemene group, ifenprodil groupNR2BandcAMPThe protein expression level was significantly reduced（P＜0.001），MOPRThe protein expression level was significantly increased（P＜0.001）。
4 discuss
Bone cancer pain is one of the main symptoms of advanced bone cancer, which is due to the severe compression, damage and stimulation of the nerve rich capsule, blood vessels, nerve fibers, etc., caused by the obvious swelling, tissue necrosis and erosion of the tumor body in the late stage of bone cancer patients, and the unbearable and intense irritating pain[11-12]。Morphine is widely used in pain treatment, especially in patients with advanced bone cancer pain, but long-term application will produce morphine tolerance and affect its analgesic effect. Its mechanism may be related toMOPRDesensitization is related to internalization, so the improvement of morphine tolerance is the focus of cancer pain treatment at present[13]。β-Elemene is an oily monomer isolated from the volatile oil of Curcuma wenyujin, a Zingiberaceae plant. It can play an anti-tumor role by regulating the proliferation, apoptosis, differentiation, metastasis and other links of tumor cells[14]In recent yearsβ-The analgesic efficacy of elemene has attracted extensive attention of researchers, butβ-The effect of elemene on the improvement of morphine tolerance in patients with bone cancer pain remains to be further studied[15]Therefore, the present study constructed a rat model of bone cancer pain-Chronic morphine tolerance model, givenβ-The results showed that with the increase of morphine administration time, the mechanical withdrawal threshold and heat withdrawal latency of rats gradually decreased, and the mechanical withdrawal threshold and heat withdrawal latency of rats were significantly decreased.The effects of ifenprodil, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, morphine, and morphine on the mechanicalβ-Elemene can effectively inhibit the decrease of mechanical withdrawal threshold and heat withdrawal latency in rats, andβ-The effect of elemene was dose related, indicating thatβ-Elemene has a certain analgesic effect and can effectively relieve morphine tolerance in rats with bone cancer pain.
In recent years, studies have found that regulating the expression level of pain related genes is an important mechanism for opioids such as morphine to exert analgesic effects[16]。MOPRIt is the most important subunit for opioids to exert analgesic effect. Opioids andMOPRWhen combined, it can effectively exert analgesic effect[17]WhenMOPRWhen bound to opioid agonists, intracellularcAMPEqual signal concentration decreases,Ca2+The inflow decreases,K+Increased efflux, leading to presynaptic membranesPThe release of substances is reduced, preventing the conduction of pain impulse, thereby exerting analgesic effect[18]。NMDAThe receptor consists of subunitsNR1、NR2、NR3Composition, whereNR2BIs the major regulatory subunit involved in pain production[19]However, under the long-term effect of opioids,MOPRfromGProtein coupled receptor kinase（G protein-coupled receptorkinases，GRKs）Catalyzes the uncoupling of phosphorylation machinery, which can activate downstream PhospholipasesC, adenylyl cyclase pathway, leading tocAMPThe expression levels of triglyceride, inositol triphosphate in the cells gradually increased, causingCa2+Increased influx activates protein kinasesC（proteinkinase C，PKC）；PKCAfter activation, it translocates to the cell membrane, while enablingNR2BActivation, further activationPKC, regulating neuronal sensitivity[20]. for further studyβ-The specific mechanism of elemene in alleviating morphine tolerance, the bone cancer pain in rats was constructed in this study-Chronic morphine tolerance model and morphine tolerance cell model, found that morphine tolerance can promoteNR2B、cAMPExpression, inhibitionMOPRExpression, administration of ifenprodil orβ-After elemene, it showed the opposite trend, andβ-The effect of elemene was dose related, indicating thatβ-Elemene can promoteMOPRExpression of, inhibition ofNR2B、cAMPIt can relieve the sensitive state of neurons, prevent the conduction of pain impulse, and then exert analgesic effect and improve morphine tolerance.
In the whole process of pathological pain generation and maintenance, neurons and glial cells are an interactive information network[21]Studies have shown that peripheral pathological pain stimuli such as bone cancer pain can make the central end of primary afferent nerve fibers and secondary neurons conducting pain release neurotransmitters to activate glial cells, and the activated glial cells releaseTNF-α、IL-1β、IL-6And other proinflammatory cytokines, further promote the release of neurotransmitters from the central end of primary afferent fibers and secondary neurons, produce and maintain pathological pain, and amplify pain[22]Other studies have shown that inflammatory response also plays an important role in morphine analgesic tolerance. In the morphine analgesic tolerance model, proinflammatory factorsTNF-α、IL-1β、IL-6The expression level was significantly increased, and blocking the production of inflammatory factors could effectively alleviate morphine resistance[23]The results of this study found that morphine tolerance could promoteTNF-α、IL-1βandIL-6 mRNAExpression, administration of ifenprodil orβ-After elemene, it showed the opposite trend, andβ-The effect of elemene was dose related, indicating thatβ-Elemene can inhibitTNF-α、IL-1βandIL-6To reduce the transmission of pain impulse, and then play an analgesic role and improve morphine tolerance.
To sum up,β-Elemene has a certain analgesic effect, which can be controlled byMOPR/NR2BIt can effectively relieve morphine tolerance in rats with bone cancer pain.
Conflict of interest
References (omitted)
Come ; [J] . Chinese herbal medicine, 2022, 53 (10): 3070-3077