Study Of Treatment Endometriosis With Thiodigalactoside

BackgroundEndometriosis(EMs)is defined as the ectopic location of the epithelial and stroma components outside the uterus cavity.It is a chronic inflammatory disorder alive with the stimulation of estrogen.Though EMs is a benign disease,it is characterized by the biologies of invasion,migration and the ability of recurrence which resemble to the malignant tumors.These features point out that same pathogenesis may exist between these two different illnesses.As indicated by many other studies,angiogenesis and inflammation definitely play an important role in the development of endometriosis.Galectin-1(Gal-1)has been widely studied in malignant neoplasms with a higher expression generally compared to the normal ones.It is distributed both intracellularly and extracellularly,involved in cell adhesion,blood vessel formation,and other physiological and pathological processes.Thiodigalactoside(TDG)is a non-metabolic inhibitor of Gal-1.It is proved that it can suppress the formation of blood vessels,improve the inflammatory state,and balance the unstable immune system.Our study aims to establish models of EMs in rats,and investigate the expression of Gal-1 in ectopic endometrium of the animal models.At the same time,the therapeutical effect of TDG on EMs is observed,and on the basis of exploring the markers of angiogenesis and inflammation,we try to clarify the underlying mechanism of TDG treatment EMs.Objective1.To set up EMs models in rats,and explore expression of Gal-1 in ectopic endometrium.2.To evaluate the therapeutical effect of TDG.3.To study the effect of TDG on angiogenesis and inflammation of the focus.4.To discover that TDG may function through Gal-1/ERK signaling pathway.5.To study the effect of TDG on gonadal hormones in rat serum.Methods1.Performing vaginal smear for Wistar rats to observe the estrous cycle at the same time every day morning.And stain the smears by Geimsa staining.2.After we observed two successive estrus cycles,we started up to establish EMs models which were surgically induced by autotransplanting endometrium onto the peritoneum in estrus of the third estrous cycle.In the sham-operated group,the left uterus horn was cut off and abandoned.After 28 days,the wound was opened again and the implants were observed.Successful criteria:(1)General criteria:the grafts on the abdominal wall grow into vesicles with clear or coffe-like liquid inside.Vascular formation was seen on the surface and the surrounding of the vesicles.(2)Pathology standard:microscopically,endometrial epithelium and matrix structure were observed in the grafts.3.Rat models were randomly divided into model group and drug group.Ectopic vesicles on the abdominal wall in model group and the endometrium of the right side in the sham-operated group were taken down.Immunohistochemistry(IHC),real-time fluorescent Quantitative polymerase chain reaction(Qrt-PCR)and Western blot(WB)for Gal-1 expression evaluation were performed respectively.Treament group was randomly divided into control group,low dose of TDG(20 mg/kg body weight)group and high dose of TDG(80 mg/kg body weight)group.PBS or TDG was intraperitoneally injected once every two days for two consecutive weeks.Then the third laparotomy was performed to assess the growth of ectopic endometrium.4.Measure and compare the size of ectopic lesions before and after treatment.Assess the expression level of Bcl-2 by WB and observe the apoptotic cells by TUNEL.5.Analyse the expression of angiopoietin-2(Ang-2)and matrix metalloproteinase-9(MMP9)by WB and detect the conceration of TNF-a in serum by Elisa.6.Analyse the level of phosphorylation of ERK by WB.7.Detect the level of gonadal hormones in rat serum.Results1.All rats had consecutive estrus cycle with a frequency of every 4-5 days for one cycle.2.Grossly,the implants of 31 rats grew into hemispheric cysts with neovascularization on the surface and clear or choclate-like fluid inside the pouch.And the implants of two rats appearanced significant fibrosis without blood vessls surrounded.Microscopicly,the cyst was consisted of epithelial cells and spindle-shaped stomal cells with microvessles.3.Microscopicly,Gal-1 was only located in the stromal layer other than in the epithelium of both groups.And in model group,Gal-1 mainly located in the cytoplasm and extracellular matrix,whereas it was mostly expressed in the nuclear in the sham group.4.Both the mRNA and protein level of Gal-1 in the model group were significantly higher than in the sham group(Both P<0.05).5.The volume did not have diffenrence between groups before treatment(P>0.05).After treatment,the volume in control group did not show markedly change.The reduction of volume in both low dosage(20mg/kg)and high dosage(80mg/kg)groups was statistically different(Both P<0.05).6.The expression of Bcl-2 decreased with the increasing of TDG conceration.There were statistical significance between both dosage groups with control group(Both P<0.01),whereas the difference between the low dosage group and high dosage group showed no statistic significance(P>0.05).TUNEL showed a higher rate of apoptic cells in high-dose group than in the control group(P<0.01).7.Compared to the control group,the protein expression level of Ang-2,MMP9 and TNF-a showed a dosage-dependent decrease.For Ang-2 and MMP9,the difference between control group and high-dose group was statistically significant(Both P<0.01).The conceration of TNF-? was lower in the high-dose group than in the control group(P<0.05).8.The expression of Gal-1 did not show significant difference between each two groups(P>0.05).Phosphorylation of ERK was reduced with the increasing of dosage conceration.However,only the difference between the control group and high-dose group had statistic significance(P<0.01).9.After 14 days treatment with PBS or TDG,the level of E2,P,LSH and LH didn't present significant difference between each two groups(P>0.05).Conclusion1.The high expression of Gal-1 in the ectopic focus makes the EMs rat an effective research model to investigate the function of this protein.2.TDG was able to reduce the volume and the expression of Bcl-2,promoting apoptosis of the ectopic focus.It may be an effective molecule to ease the development of EMs.3.TDG reduced the expression of Ang-2,MMP9 and TNF-?,and showed an inhibitory effect on angiogenesis and inflammation.4.TDG may be able to occupy the carbohydrate recognition domain of Gal-1 to inhibit its function and the phosphorylation of ERK was hampered.5.The level of gonadal hormones were not affected by TDG,so it may not inhibit the hypothalamus-hypophysis-gonad axis.