Effect Of Botulinum Toxin A On Endometriosis-associated Pain And Its Molecular Mechanisms In Mice

ObjectivesEndometriosis is a chronic gynecological pain disease which is caused by endometrial cells planted in peritoneal cavity unexpectedly with incidence of 10%in women of childbearing age.Surgical resection is still the main treatment for endometriosis.There are no rapid and effective therapies for endometriosis and patients need long-term pharmacotherapy after surgery.Neuroinflammation is one of the major reasons for initiation and maintenance of chronic gynecological pain.Activated neurogliocyte plays an important role in pain initiation and transduction.Previous studies showed that expressions of P2X,P2Y and CXCR receptor families were higher in activated spinal microglia which upregμl ated cells secretory activities.Inflammatory factors,IL-1β,IL-6,TNFαand PGE2,of depolarized and excited neurons lead to central sensitization and hyperalgesiaultimately.Notably,PI3K/Akt signaling pathway was up-regulated in spinal microglia during the process of neuropathic and inflammatory pain.Mechanical withdrawal threshold can be relieved by blocking PI3K/Akt signaling pathway.Botulinum toxin type A（BoNT-A）,as a kind of cell toxins,is produced in Clostridium botulinum and can bind to the presynaptic nerve terminals and enter cells to inhibit release a synaptic vesicles and block the signal transduction.Animal studies have shown that BoNT-A could inhibit the release of pain mediators,thereby reducing the pain caused by the drug’s inflammatory response.In vivo,BoNT-A has been shown to inhibit the pain mediators release in damaged bladder tissue,such as bradykinin,substance P,as well as inhibit the secretion of calcitonin gene-related peptide and glutamate in dorsal root ganglia and trigeminal ganglia,attenuating central sensitization.BoNT-A could inhibit the secretion and release of a variety of neurotransmitters,such as glutamate and substance P in early gene expression period in dorsal root,under the harmful stimulus conditions or peripheral nerve injury to block central and peripheral sensitization.BoNT-A has been widely used in pathological pain clinically,such as migraine,muscul oskeletal pain and refractory prosopalgia.However,it has never been reported that BoNT-A was used in treatment of endometriosis pain,based on the vital role in neuropathtic pain,we suppose that BoNT-A could act as an analgesic chemical in pain treatment.This study focuses on the mechanism and potential applied value of BoNT-A on neuropathic pain of endometriosis by ethology and molecular methods.Materials and methods1.Effect of Bo NT-A on morphology change of BV-2 cells induced by LPS treatmentMouse microglia BV-2 were cultured in DMEM+10%FBS+1%Gln+1%P/S in37oC,5%CO₂ constant temperature incubator.Cells were divided into plates by 1:3when concentration grows to 80%.After 24 hours,LPS,BoNT-A,LPS+BoNT-A and solventia were added into plates for incubating another 24 hours.Cells morphology was identified under microscope.2.Effect of BoNT-A on inflammatory factors release and protein expression of BV-2cells induced by LPSAfter LPS,Bo NT-A,LPS+BoNT-A and solventia treatment for 24 hours,expression of Iba1 and OX42 in cells was observed by laser scanning confocal microscope.IL-1β,IL-6,TNFαand PGE2 observed in cellular supernatant were evaluated by ELISA test.The mRNAs and the protein expression of P2X7,CX3CR1,Akt,P38 MAPKand JNK were evaluated by qPCR and Western blot,individually.3.Endometriosis modeling（EMs）establishmentFifty-five 15-16 g BALB/c mouse were adapted raised for 4 weeks to reach 20-22g per mouse.Fifteen fat mice were selected to be injected estrogen（in estradiol benzoate）with 100μg/kg twice.Uteruses were dissected and grinded into sterile saline after one week.The uteruses pieces were injected into enterocoelia.Oxytocin was injected into model mice enterocoelia with 20 IU/kg by NO.26 syringe needle.Obvious writhing response indicates the success of modeling.4.The therapeutic effect of BoNT-A on writhing response in EMs miceThe writhing reactions were tested at day 0,1,3,5 and 7 after BoNT-A or control drug administration.Data and the therapeutic effects were analyzed.5.Effect of Bo NT-A on activated microglia cells resides in spinal cord in EMs miceThe lumbar enlargement of spinal cord was taken out after 7 days for spinal cord tissue section preparation.Iba1 and OX42 protein expression were evaluated by laser scanning confocal microscope.The lumbar enlargement of spinal cord was taken out after 7 days for RNA and protein extract to evaluate P2X7,CX3CR1,Akt,JNK,P38MAPK,p-Akt,p-JNK and p-P38MAPK gene expression.And spinal cord tissue sections were prepared to evaluate P2X7 and CX3CR1 protein expression by immunohistochemistry.Results1.Morphology change of BV-2 cells induced by LPS treatmentThe morphology of BV-2 cells was changed into spindle-shape from roundness after LPS stimulation.The 1μg/ml concentration of LPS was selected for subsequent experiments.2.LPS induces the release of inflammatory factors and the change of protein expression in BV-2 cellsConfocal microscopy observation showed that the protein expressions of Iba1 and OX42 were up-regulated after LPS treatment for 24 hours.The secretions of IL-1β,IL-6,TNFαand PGE2 were up-regulated after LPS stimulation.3.BoNT-A inhibit BV-2 activation and inflammatory factors releaseThe levels of proteins and inflammatory factors mentioned above decreased after BoNT-A treatment.The JNK gene expression showed same consequences.4.Microglia have been activated in spinal cord in EMs miceOn Day 1,3,5 and 7 after drug administration,the writhing reaction of model mouse was significantly enhanced.In EMs group,Iba1 and OX42 proteins expression were higher than BoNT-A group evaluated by laser scanning confocal microscope.Compared with the sham group,the protein expression of P2X7,CX3CR1,Akt,JNK,P38MAPK,p-Akt,p-JNK and p-P38MAPK were increased significantly in EMS group.The expression of P2X7 and CX3CR1 rises firstly and then decreases pre-and post-treatment evaluated by immunohistochemistry in EMs group.5.BoNT-A attenuate the endometriosis-associated pain by affecting the expression of P2X7,CX3CR1,Akt and the phosphorylation of P38MAPK,JNKThe writhing reaction of model mouse was significantly attenuated in BoNT-A group mice,so as the Iba1 and OX42 proteins expression examined by confocal microscope.Compared with the surgical group,the expression of P2X7,CX3CR1,Akt,JNK,P38MAPK,p-Akt,p-JNK and p-P38MAPK protein were down-regulated in BoNT-A mice group.ConclusionsBoNT-A suppressed in vitro BV-2 cells activation and the secretion of inflammatory factors.However,the molecular mechanism of BoNT-A inhibition on LPS-induced microglia activation is unclear.BoNT-A decreased in vivo intensity of writhing reaction and the protein expression of P2X7,CX3CR1,Akt,JNK,P38MAPK,p-Akt,p-JNK and p-P38MAPK in endometriosis mouse model.BoNT-A may play a promising role in relieving endometriosis pain by down-regulation of Akt,JNK and P38MAPK in spinal microglia.Therefore,BoNT-A may have potential value in the treatment of endometriosis pain,which is worthy of further study.