| Background and AimsPain can be divided into inflammatory pain(also called nociceptive pain)and neuropathic pain,inflammatory pain comes from natural irritation of damaged tissue(surgery,osteoarthritis or trauma)or chemical mediators;Neuropathic pain is caused by direct damage of sensory nerves or related diseases(diabetes or shingles).And this pain is mainly caused by changes in ion channels that produce action potentials in the nerves,which alter electrophysiological events.A special type of pain-the pain induced by cancer may involve inflammation and neuropathology.On the one hand,tumor metastasis induces tissue damage and releases various inflammatory mediators,such as TNFa and IL-6.On the other hand,tumor can also grow inside sensory nerves or compress the nerves of target tissues,which can cause neuropathic changes.Cancer pain can be thought of as a combination of pain,but there is a large amount of evidence indicating that it has other unique characteristics,which means it should be treated as a unique pain state.Among all kinds of cancer pain,bone cancer pain(BCP)caused by bone metastases is an extremely severe type of pain.Bone cancer pain is the most common type of moderate and severe cancer pain,and approximately 75%of patients with advanced cancer experience bone cancer pain,which severely affect their quality of life.The World Health Organization(WHO)advocates that the analgesics remain the main therapeutic approach for the treatment of bone cancer pain in clinical,such as opioids(morphine,oxycodone,and hydromorphone).The main site of action of opioids is the spinal cord,which achieves analgesia by attenuating the sensitivity of peripheral receptors,reducing the input of pain,and inhibiting the activity of spinal cord neurons.In addition,bisphosphonates,radiotherapy,and chemotherapy drugs also play a role in the treatment of bone cancer pain.At present,conventional treatment of bone cancer pain usually requires the combination of multiple treatment methods.For example,radiation therapy was applied to the site of pain or bone metastases with systemic treatment using opioids or other drugs.In some special cases,the use of radioisotopes and non-invasive techniques may contribute to the treatment of metastatic bone cancer pain.However,BCP is still not fully controlled,although some patients fully follow WHO guidelines for analgesia.One of the reasons for inadequate pain management is opioid-related adverse reactions,including constipation,sedation,itching,nausea and as well as vomiting.Other reasons for inadequate pain management may be fear of drug addiction,physical dependence,and insufficient effectiveness of analgesics.Moreover,chemotherapy or radiotherapy itself has a very high requirement for the patient's physical condition,and during the course of treatment they will also induce a series of adverse effects.Although neurons have long been thought to play a crucial role in the development and maintenance of bone cancer pain,a large amount of emerging evidence suggests that glial cells activation can also play a similar role.Glial cells account for about half of the entire volume of brain and spinal cord.It can be divided into astrocytes,microglia,and oligodendrocytes.As the most abundant glial cells in the central nervous system,astrocytes can eliminate excess ions and recycle secreted neurotransmitters when synaptic transmission regulates the external environment of neurons.Microglia are resident macrophages within the central nervous system,which protect neurons by engulfing infected or damaged cells.Under physiological conditions,both astrocytes and microglia are in a quiescent state.However,they can be activated when damage occurs.In rodent bone cancer pain models,the number of astrocytes and microglia increases significantly in the dorsal horn of the ipsilateral spinal cord.In addition,inhibition of glial cells activation and pro-inflammatory cytokines released by glial cells can relieve BCP-associated mechanical allodynia,thermal hyperalgesia,and spontaneous pain.In addition,the injection of activated microglia in the sheath of naive rodents can reduce mechanical pain.These studies indicate that glial cells play a key role in bone cancer pain.Many of the cytokines involved in the development of bone cancer pain are secreted after glial activation.Tumor necrosis factor-alpha(TNF-alpha)is a pro-inflammatory cytokine that is mainly produced by activated macrophages and some specific tumor cells.It has been reported that TNF-? may promote angiogenesis,induce tumor growth and promote tumor metastasis through different signal transduction pathways.In addition,mechanical hypersensitivity was induced after foot-injection of TNF-? in naive and fibrosarcoma mice,suggesting that TNF-? may be able to induce peripheral sensitization.Moreover,there is a lot of evidence that the spinal cord glial cell-derived pro-inflammatory cytokines are significantly elevated in the bone cancer pain model,such as interleukin-1?(IL-1?),interleukin-6(IL-6),and TNF-?,which participate in the pathogenesis of chronic pain and mediate pain behavior.By enhancing spinal excitatory synaptic transmission and promoting pain states,TNF-? and IL-1? can directly induce central sensitization.These studies have also demonstrated that inhibition of these pro-inflammatory cytokines can inhibit the activity of glial cells,thereby reduce bone cancer-induced mechanical allodynia and spontaneous pain,which can be evaluated by paw withdrawal threshold(PWT)and rating of dynamic pain.These observations in laboratory animals suggest that inhibiting inflammatory reactions and glial cell activation could be potential strategies for chronic pain treatment.Indeed,direct injections of inhibitors for glial cell activation into the spinal cord could attenuate allodynia and hyperalgesia.3,5,7,2',4'-pentahydroxyflavone(Morin)is a natural bioflavonoid,which is found in many herbs and fruits.It was originally isolated from members of the Moraceae family.It can be extracted from the leaves,fruits,stems and the branches of many plants.Morin has been used as food and herbal medicine,without toxicity or side effects.Some studies have shown that Morin may be beneficial to several human diseases.In fact,Morin exerts antioxidant,anti-diabetic,anti-inflammatory,anti-tumor,anti-hypertensive,anti-bacterial,and neuroprotective effects by regulating the activity of many enzymes.For example,Morin protects muscle cells,endothelial cells,liver cells,and red blood cells by antioxidation;prevents nerve degeneration by inhibiting nerve inflammation;and induces apoptosis to prevent cancer.In some cases,Morin showed systemic protection that can reduce the side effects of several drugs without influencing its normal function.More and more studies has showed that Morin can regulate cell signaling pathways related to different chronic pathophysiological conditions,including gastrointestinal complications,diabetes,cardiovascular diseases,cancer,arthritis,neurodegenerative diseases and several other inflammation diseases.Because various oxidative stress-related diseases are caused by inflammation,the anti-oxidant and anti-inflammatory activities of Morin play a key role in the treatment of these diseases.Different studies have proven that Morin is very effective in chronic diseases,such as chronic neurodegenerative diseases,chronic cardiovascular diseases,chronic gastrointestinal diseases,chronic hypersensitivity and immune disorders,cancer and several chronic pathologies associated with oxidative stress physiological disorders,arthritis,diabetes.Morin changes the levels of phosphorylated Akt kinase,Erkl/2,AIF release,cytoplasmic Bax levels,and regulates nuclear translocation of NF-?B,and prevents the imbalance caused by the different levels and activities of different enzymes(eg,elastase),pro-inflammatory mediators,and pro-inflammatory cells,inflammatory enzymes,glycoproteins,reactive oxygen species,lysosomal acid hydrolase,and transcription factors(such as NF-?B,p65,and AP1),and which will achieve optimal therapeutic effect.In addition,both in vitro and in vivo studies have shown that Morin exhibits very low levels of toxicity and its long-term application is well tolerated.All these findings indicate that Morin can be used alone or in combination with other drugs to prevent many human diseases.Because glial cells activation and inflammatory response play an important role in the development of bone cancer pain.Morin has a good effect on the treatment of chronic diseases,which can exerts anti-inflammatory,anti-tumor and neuroprotective effects.At present,we still do not know the role of Morin in bone cancer pain and how it regulates bone cancer pain.And there is no good therapeutic approach for bone cancer pain,so we are urgently need to explore the role of Morin in bone cancer pain and the mechanisms by which it exerts analgesia.And we wonder if Morin can be a potential drug for the mangement of bone cancer pain.Materials and MethodsUnilateral tibia bone cancer rat model was established as described following.Briefly,rats(160-180 g)were deeply anesthetized with intraperitoneal injection of 50 mg/kg pentobarbitol sodium(1%in saline).Arthrotomy was performed to expose left tibia.5 ?l Walker 256 rat mammary gland carcinoma cells(1×105/?l)were injected into the left tibia cavity.Sham-operated rats were received 5 ?l of normal saline treatment.To prevent leakage of cells,the injection needles were kept in place for 2 mins after finishing injection.The injection site was sealed with bone wax and muscle and skin layers were sutured separately.Rats were injected with Morin at indicated doses(5 mg/kg,10 mg/kg and 20 mg/kg)for consecutive 7 days once daily.Pain behavior was characterized by scores of paw withdrawal thresholds(PWT)and Ambulatory pain The sensitivity of the hind paw to tactile allodynia stimulated by Von Frey filaments was measured using a Von Frey filament measurement instrument.Ambulatory pain was measured from hind limb use during normal ambulation in an open field.Normal limb use during spontaneous ambulation was scored on a scale of 0 to 4:(0)normal use,(1)some limping,but not pronounced,(2)pronounced limping,(3)pronounced limp with guarding behavior,(4)complete lack of use of the limb.All behavioral tests were performed in a double-blind manner until examiners need to decode data for analysis.Real-time RT-PCR and Western blot(WB)methods were used to detect the mRNA and protein expression levels of GFAP,TNF-?,IL-1?,IL-6,IL-10 and CB2.In additon,the protein bands were analyzed quantitatively.Data were presented as meanąSD.One-way or two-way ANOVA analysis followed by a Tukey's post hoc test was performed.p value less than 0.05 was considered significant.The analysis was carried out using SPSS software(SPSS).ResultsWe found that rats with cancer developed and maintained mechanical allodynia.And the paw withdrawal threshold in rats bearing cancer was different from the paw withdrawal threshold in the sham group which was more and more higher after the fourth day after surgery.There was a declining trend on 7,10,13 and 16 days in the paw withdrawal threshold in rats bearing cancer.Furthermore,the rats in the bone cancer group also developed ambulatory pain behaviors.The ambulatory pain score of rats in the bone cancer group were getting higher and higher at the 7th,10th,13th,16th after the surgery.In addition,we found that Morin treatment from the 7th day after the surgery resulted in significantly higher withdrawal paws thresholds than untreated rats bearing cancer,and the ambulatory pain score was also significantly lower than that of untreated cancer rats.Morin reduced mechanical allodynia in cancer bearing rats in a dose-dependent manner,compared with untreated rats.Morin was also found to dose-dependently attenuate free movement pain in cancer bearing rats(5 mg/kg,10 mg/kg,20 mg/kg).Therefore,the relief of pain behavior was most pronounced when the Morin dose was 20 mg/kg.Because the activation of astrocytes plays an important role in the bone cancer pain model,we examined the activation of astrocytes in an established bone cancer pain rat model.The results of qPCR and western blot showed that both mRNA and protein levels of GFAP,a marker of spinal cord astrocyte activation,were significantly up-regulated in the bone cnacer pain group compared with the sham group.When rats bearing cancer were treated with Morin(20 mg/kg),the expression levels of GFAP were significantly down-regulated compared with the untreated cancer group.Therefore,the treatment of Morin inhibits the activation of spinal astrocytes in cancer pain models.Up-regulation of pro-inflammatory cytokines is often associated with glial activation and is widely involved in the development of bone cancer pain.The detection of pro-inflammatory cytokines and anti-inflammatory cytokines can be found in the bone cancer pain model group.We found that the mRNA and the protein levels of pro-inflammatory cytokine TNF-?,IL-1? and IL-6 and the anti-inflammatory cytokine IL-10 were significantly increased on the 7th day after the implantation of tumor cells to rats.However,after treatment with Morin(20mg/kg),the mRNA and protein levels of pro-inflammatory cytokines TNF-?,IL-1? and IL-6 were significantly down-regulated,while the mRNA and protein levels of anti-inflammatory cytokine IL-10 were significant increased.These results indicate that Morin can differentially regulate the expression of pro-inflammatory and anti-inflammatory cytokines in the spinal cord of a rat model of bone cancer pain.To further elucidate the mechanism of the analgesic effect of Morin in a rat model of bone cancer pain,we injected intrathecally AM630(a selective cannabinoid CB2 receptor antagonist)or injected intraperitoneally naloxone(an opioid receptor antagonist)before Morin injection.The results showed that pretreatment with naloxone(1 mg/kg)has no effect,while pretreatment with AM630 attenuated Morin's anti-nociception effects.The paw withdrawal threshold on the 7th day after the surgery was significantly lower than that of the Morin treatment group after AM630 pretreatment.And there was no significant difference compared with the bone cancer pain group.On the 16th day after the surgery,compared with the Morin treatment group,there was no difference in the mechanical threshold of AM630 pretreatment.The paw withdrawal threshold of naloxone pretreatment was significantly higher than that of the bone cancer pain group from the 7th day to the 16th day after the surgery,but there was no significant difference from the Morin treatment group.The same result was also found in the score of ambulatory pain.Moreover,we found that increasing the dose of naloxone(10 mg/kg,40 mg/kg,80 mg/kg)by intrathecal injection still failed to block the analgesic effect of Morin.In addition,we also found that the protein expression level of CB2 in the bone cancer pain group was significantly decreased compared with the sham group.After treatment with Morin(20 mg/kg),the protein expression level of CB2 was significantly up-regulated,although it was still lower than the sham group.The above results indicate that activation of CB2 receptor may mediate the analgesic effect of Morin in a rat model of bone cancer pain.ConclusionsRats with bone cancer can produce and maintain mechanical allodynia and ambulatory pain behavior.This pain-related behavior can be alleviated after Morin treatment,and the relief of this pain-related behavior will increase in a dose-dependent manner of Morin.When the dose of Morin is 20 mg/kg,the analgesic effect achieved is most obvious.We also found that Morin(20 mg/kg)significantly reduced the expression of GFAP up-regulated in rats bearing cancer,indicating that Morin can inhibit the activation of astrocytes.Moreover,Morin can differentially regulate the expression of pro-inflammatory cytokines and anti-inflammatory cytokines.Morin can block the increase of the pro-inflammatory cytokines TNF-a,IL-1?,and IL-6 caused by cancer and up-regulate the expression of the anti-inflammatory cytokine IL-10.In addition,pretreatment with AM630 attenuated the anti-nociception effects of Morin,while pretreatment with naloxone(1mg/kg)has no effect.And the protein levels of CB2 receptor increased dramatically after Morin treatment,indicating that CB2 receptor may mediate Morin analgesic effect in a rat model of bone cancer pain.In summary,natural plant-derived Morin can exert anti-nociception effects by inhibiting the activation of neural astrocytes and the occurrence of neuroinflammation,which may be mediated by the activation of CB2 receptor.Therefore,Morin is expected to be a safe and effective drug for the mangement of bone cancer pain. |
PDF Full Text Request