Ginsenoside Rf Alleviates Dysmenorrhea And Inflammation Through The BDNF-TrkB-CREB Pathway In A Rat Model Of Endometriosis

Part ? Ginsenoside Rf reduces the endometriosis volume,alleviates dysmenorrhea and inflammation in endometriosis rat modelBackground:Endometriosis is an estrogen-dependent inflammatory disease in which the functional endometrial tissue(endometrium)grows outside the uterine cavity and it can affect approximately 10%of the female population in the reproductive age.The major symptoms of endometriosis are infertility and endometriosis-related pain.Dysmenorrhea,chronic pelvic pain,dyspareunia,and dysuria are the main symptoms of endometriosis-related pain,which is really hard to endure.Some of them often induce dysfunction such as urinate etc.Surgery and hormone therapy are the dominant treatments for endometriosis.Surgerys often relate to recurrence.Recurrence rate is about 50%in 5-year after surgery.The recurrence of pain often precedes the discovery of the lesion.Hormone therapy often causes serious treatment-associated side effects,such as hepatic injury and osteoporosis,which can make it worse.Sometimes drugs will become ineffective with the gradual aggravation of pain.So,it is urgent to develop a new therapeutic strategy for the treatment of endometriosis.Endometriosis pain is due to interaction of central and peripheral mechanisms.Central sensitization,synaptic plasticity and long-term potentiation are included in the central mechanisms.Peripheral mechanisms are related to mechanical stimulation on nerve endings by enlarged pression in lesions,chemical stimulation on nerve endings due to inflammatory factors and iron ions,and imbalance distribution of sensory and sympathetic nerves in lesions.Inflammation plays a key role in the occurrence and deterioration of endometriosis pain.Flammatory cytokines can enhance the proliferation,invasion and adhesion of endometrial cells,promote angiogenesis,and contribute to the growth and maintenance of endometriosis.They can also induce the nerve sprouting,stimulate nerve fibers and further sensitize nerve endings,which not only induces pain,but also further leads to the pain deterioration.A thorough understanding of the mechanism can help us find a more effective treatment for endometriosis pain.Ginsenoside Rf,a glycoside of protopanaxatriol from Panax ginseng,has been traditionally used in herbal medicine in both Asian and Western countries.Ginsenoside Rf has been testified to have an antinociceptive effect in some in vivo and in vitro models.Recently,ginsenoside Rf has been shown to have some anti-inflammatory effect by down-regulating the production of some pro inflammatory cytokines such as IL-6?IL-1? and inhibiting the activation of inflammation associated pathways,which may play a key role in the development of endometriosis.Therefore,we speculate that ginsenoside Rf may alleviat pain and inflammation,inhibit growth and swelling in endometriosis.No investigation has been designed to test the effects of ginsenoside Rf on rat endometriosis-associated dysmenorrhea.Thus,this study is designed to test whether ginsenoside Rf systemic treatment has antinociceptive and anti-inflammatory effects in the rat endometriosis model.Objective:A surgically induced rat model of abdominal endometriosis was established to investigate the effects of ginsenoside Rf on endometriosis pain and inflammation.Based on the understanding of the pain mechanism of endometriosis,the effects of different concentrations of ginsenoside Rf were tested in three aspects:lesion volume,pain response and inflammation response.Methods:Endometriosis model was constructed through uterine horns homologous transplantation.Following a further recovery period of 2 weeks,the Wistar rats were assigned to 6 groups:the sham group,normal control rats(n-16);the estradiol valerate(E2V)control group,normal rats treated with E2V(0.5 mg kg-1)daily for 14 consecutive days to induce dysmenorrhea(n=8);the endometriosis group,rats with endometriosis treated with E2V(0.5 mg kg-1)daily for 14 consecutive days and 0.9%saline vehicle during the last 7 days(n=13);and the three ginsenoside Rf groups(Rf 1,Rf 2,and Rf 4),rats with endometriosis treated with E2V(0.5 mg kg-1)daily for 14 consecutive days intraperitoneally injected with ginsenoside Rf(1.0 mg kg-1,2.0 mg kg-1,and 4.0 mg kg-1,respectively)during the last 7 days(n=1 5,16,and 14 for each group).On day 14,an intraperitoneal injection of oxytocin(2U per rat)was administered to induce writhing response one hour after the last administration of E2V.Half an hour after the observation of writhing responses,the ectopic endometrial tissues were measured and resected.(1)The writhing frequency,duration of an abdominal writhe and writhing latency were used to measure dysmenorrhea in the rats with endometriosis.(2)The ectopic endometrial tissues were measured and resected.An ellipsoid volume formula(?/6×length×width×height)was adopted.(3)Quantitative real-time RT-PCR was used to detecte the mRNA expression of inflammatory cytokines including iNOS?IL-6?IL-1B?TNF-a.(4)Elisa was used to detecte the expression of VEGF in peritoneal fluid.(5)Quantitative real-time RT-PCR was used to detecte the mRNA expression of VEGF in ectopic endometrial tissues.Results:(1)A statistically significant reduction in the implant volume was shown in the ginsenoside Rf-treated groups in a dose-dependent manner compared with the model group.(2)Ginsenoside Rf ameliorates dysmenorrhea in the rats with endometriosis.The endometriosis model showed a nearly 2.5-fold increase in writhing frequency and a 2.1-fold increase in the duration of an abdominal writhe when compared with the E2V rats,which could be reduced by ginsenoside Rf following a 7-day treatment in a dose-dependent manner.The endometriosis model showed decreased writhing latency from 7 minutes to 2 minutes as compared to the E2V rats and a 7-day treatment with ginsenoside Rf could lengthen the writhing latency in a dose-dependent manner.(3)Ginsenoside Rf can inhibit the inflammatory response of endometriosis in rats.The mRNA expression of iNOS,IL-6,IL-1?,TNF-a in model group was significantly higher than that in sham group.the relative expression of the inflammatory molecules iNOS,IL-6,IL-1? and TNF-? was significantly down-regulated by ginsenoside Rf in the ectopic implants.The greater the concentration of the drug,the more obvious the inhibition is.(4)Ginsenoside Rf inhibits the expression of VEGF in endometriosis.As an important neuro-angiogenic factor,the VEGF level in the peritoneal fluid increased significantly in the endometriosis group when compared with the sham group,while ginsenoside Rf could attenuate the increased levels of VEGF in a dose-dependent manner.Such a trend could also be observed in the ectopic endometrial tissues when VEGF mRNA expression was analyzed by the RT-PCR assay.Conclusions:The study reveals that ginsenoside Rf significantly reduces the size of the endometrial explants in a rat endometriosis model and alleviates dysmenorrhea and inflammation.Part II BDNF-TrkB-CREB pathway as the key target in the mechanism of which alleviates dysmenorrheal and inflammation in endometriosis rat modelBackground:Endometriosis is a common disease in women of childbearing age,with complex clinical manifestations for its different planting site.The mechanism of endometriosis is still unknow.Pain is main syndrome of endometriosis.And the main treatment of endometriosis pain is operation and hormone therapy.The problems were recurancy and side effect.Ginsenoside Rf has been testified to have antinociceptive effect and anti-inflammatory effect in some in vivo and in vitro models.It can down-regulate the expression of pro inflammatory cytokines such as IL-6?IL-1?.And those pro inflammatory cytokine also participate in endometriosis inflammation.We have exhibited the effect of Ginsenoside Rf in size of ectopic lesion,dysmenorrhea and inflammation,and we wil further discuss the mechanism of antinociceptive and anti-inflammatory effects of Ginsenoside Rf.More and more studies have shown that brain-derived neurotrophic factor(BDNF)is involved in pain regulation.BDNF is a member of the neurotrophic factor family and plays an important role in the growth,differentiation and survival of neurons.It specifically binds to tyrosine kinase receptor B(TrkB),a specific ligand,and plays various physiological and pathological roles.Increased serum levels of BDNF protein was found in patients with acute and chronic pain.Mice injected with BDNF intrathecally showed obvious hyperalgesia.These evidences suggest that BDNF is involved in central sensitization of pain.In addition,the increased expression of BDNF was found in patients with various acute and chronic inflammations,such as osteoarthritis and masseteritis,accompanied by increased excitability of neurons in the corresponding ganglion.These indicate that BDNF can sensitilize the nerve endings and neurons to participate in inflammatory pain.BDNF can also regulate inflammation.It can regulate inflammation of stroke and reduce local ischemia-reperfusion injury.Specific binding of BDNF to TrkB activates downstream ras-MAPK pathway,which further phosphorylates downstream cyclic adenosine phosphate effector binding protein(CREB)and regulates the expression of proinflammatory factors,such as NF-?B.NF-?B further regulates the expression of inflammatory factors such as IL-6 and IL-1?.Such inflammatory factors also have a role in the development of endometriosis.We designed the following experiments to observe the effect on BDNF-TrkB-CREB signaling pathway of ginsenoside Rf.Objective:To explore the mechanism of ginsenoside Rf on endometriosis pain and inflammation.Methods:As exhibited in part one,rats were grouped and treated with operation and E2V,and then were sacrified.Ectopic endometrial tissues and peritoneal fluid were reserved for following test:(1)Elisa was used to detecte the expression of BDNF in peritoneal fluid and serum.(2)Quantitative real-time RT-PCR was used to detecte the mRNA expression of BDNF,TrkB and CERB in ectopic endometrial tissues.(3)Weston blot was used to detecte the protein expression of BDNF,TrkB and pCERB.Results:(1)Ginsenoside Rf decreased the level of BDNF protein in serum and peritoneal fluid of endometriosis rats.The concentration of BDNF protein in serum and peritoneal lavage fluid in model group was significantly higher than that in sham group.Ginsenoside Rf could decrease the level of BDNF protein in peritoneal fluid and serum elevated by endometriosis in does-dependent manner.(2)Ginsenoside Rf could down-regulate the mRNA expression of BDNF,TrkB and CREB in ectopic endometrial tissues.The results showed that the expression of BDNF,TrkB and CREB in model group was significantly higher than that in sham group.The expression of BDNF was nearly three times higher than that in sham group.The expression of TrkB was about three times more than that in sham group.The expression of CREB was about 2.5 times higher than that in sham group.The difference was statistically significant.Ginsenoside Rf can reverse the over expression of BDNF,TrkB and CREB in endometriosis.Compared with model group,the mRNA expression of BDNF,TrkB and CREB were down-regulated by Ginsenoside Rf in does-dependent manner.(3)Ginsenoside Rf could down-regulated the protein expression of BDNF,TrkB and pCREB in ectopic endometrial tissues.The results showed that the protein expression of BDNF,TrkB and pCREB in endometriosis lesions of model group was significantly higher than that of sham group.The expression of BDNF protein was nearly three times higher than that of sham group,while the expression of TrkB protein was about 2.5 times higher than that of sham group.The expression of p-CREB protein was about 2.75 times higher than that of sham group.Ginsenoside Rf could down-regulate the protein expression of BDNF,TrkB and pCREB in ectopic endometrial tissues in does-dependent manner.Conclusions:Ginsenoside Rf reduces the size of the endometrial explants and alleviates dysmenorrhea and inflammation through the BDNF-TrkB-CREB pathway.It provides a new experimental basis for the treatment of endometriosis.