| Objective:Prolactinoma is harmful to human health,and its pathogenesis is still unclear.Bromocriptine is the only drug on the market in China,and it is prone to drug resistance currently.In addition,the incidence in women is much higher than in men,especially when they are young,which may be related to estrogen levels.Therefore,it is of great significance to reveal the pathogenesis of prolactin tumor and develop new treatment methods.In this study,the role of TLR4 in the process of estradiol-induced prolactin tumor was explored on the basis of previous studies,and the target of inhibiting prolactin tumor was sought by targeting the TLR4/p38MAPK pathway.Methods:The expression of ERβ,TLR4 and PRL in C57BL/6 mice and human prolactin tumor pituitary specimen were detected by immunofluorescence or immunohistochemistry.In this study,estradiol-induced C57BL/6 wild-type（WT）mouse model and TLR4-/-mouse model were used to study the role of TLR4 in prolactin tumor.The expression of ERβ,TLR4 and PRL in MMQ cells was studied by transfecting MMQ cells with estradiol,fulvestrant,lipopolysaccharide or si TLR4.Immunoprecipitation analysis was used to study the interaction between ERβand TLR4.TLR4 inhibitor TAK242 was used to study the pharmacodynamic effects of estradiol-induced prolactinoma in mice.Preparation of liposome of TAK242.（1）Pituitary specimens with prolactinoma and other pituitary diseases were collected from patients,paraffin specimens were prepared,and the expressions of ERβ,TLR4 and PRL in the samples were detected by immunohistochemistry,and the correlation between ERβand TLR4 was detected by full scan histochemical score.The expression and co-localization of TLR4 and PRL in PRL（+）and PRL（-）tissues were detected by immunofluorescence.（2）MMQ cells were induced with different concentrations of estradiol and fulvestrant,and appropriate intervention concentrations of estradiol and fulvestrant were selected.Estradiol and fulvestrant concentrations were selected to intervene MMQ at different time to determine the appropriate intervention time.The expression of PRL and ER/TLR4/p38MAPK pathway was detected by the intervention of MMQ with estradiol after blocking estrogen receptors with fulvestrant.si TLR4 was used to intervene MMQ to detect the expression of PRL and ER/TLR4/p38MAPK pathway,while estradiol and lipopolysaccharide were used to intervene MMQ to detect the expression of PRL and ER/TLR4/p38MAPK pathway.（3）Preparation of estrogen-induced TLR4-/-mouse model to study the effect of TLR4 knockout on prolactinoma.The mice were divided into four groups:WT group;ES group;TLR4-/-group;TLR4-/-ES group.Modeling method:Model group was intraperitoneally injected estradiol once every 4 days for 10 times in total,with a single dose of 20mg/kg.After modeling,serum of each group was collected for ELISA to measure serum PRL level,and pituitary tissues of mice were weighed.Three samples from each group were selected to extract total protein,and the indexes of ER/TLR4/p38MAPK pathway and PRL expression were determined by Western blot.Three samples from each group were embedded in paraffin,and the expression and localization of ERβ,TLR4 and PRL were determined by immunohistochemistry and immunofluorescence.（4）To prepare estrogen-induced prolactinoma mouse model and study the pharmacodynamic effect of TLR4 inhibitor TAK242 on prolactinoma.The mice were divided into 5 groups:control group;Model group;Bromocriptine formation;TAK242 group（0.15 mg/kg）;TAK242 group（0.3 mg/kg）.Modeling method:Model group was intraperitoneally injected estradiol benzoate once every 4 days for 10 times in total,with a single dose of 20mg/kg.After modeling,bromocriptine group was given 1.6mg/kg bromocriptine once a day for a month.TAK242 group was intraperitoneally injected with TAK242（0.15mg/kg or0.3mg/kg）twice a week for four weeks.After administration,serum of mice in each group was collected for ELISA determination of serum PRL level,and pituitary tissues of mice were weighed.Three samples from each group were selected to extract total protein,and the indices of TLR4/P38 MAPK pathway and PRL expression were determined by Western blot.Three samples from each group were paraffin-embedded,and the expression and localization of TLR4and PRL were determined by immunohistochemistry and immunofluorescence.（5）TAK242 liposome was prepared by thin film dispersion method.encapsulation rate was used as the index,drug lipid ratio,phosphorus bile ratio and ultrasonic power were selected by single factor analysis.The optimal prescription conditions of TAK242 liposome were screened.Results（1）immunohistochemical results of human pituitary specimens showed that the expression of ERβand TLR4 in pituitary tumor tissues was significantly higher than that in the negative control group,and there was a strong positive correlation between the expression of ERβand TLR4.At the same time,immunofluorescence results showed that TLR4 and PRL had a strong co-location and co-expression.（2）Different concentrations of E2 and Ful could regulate the expression of PRL and ERβ/TLR4/p38MAPK pathway proteins in MMQ cells for 48h,and 100n M OF E2 could down-regulate the expression of PRL and ERβ/TLR4/p38MAPK pathway proteins in MMQ cells.After blocking ER receptor with Ful in advance,PRL and ERβ/TLR4/p38MAPK pathway protein expression were not up-regulated after E2 administration.After TLR4 knockdown,the expression of ERβ/TLR4/P38 MAPK pathway and PRL protein were down-regulated.The up-regulation of ERβ/TLR4/p38 MAPK pathway and PRL protein was more obvious in E2+LPS group than in single ERβor TLR4.（3）The mouse model of estrogen induced prolactinoma was established successfully.Weighing results showed that compared with WT mice,the pituitary weight/body weight of ES mice was significantly increased,while the pituitary weight/body weight of TLR4-/-mice injected with ES significantly decreased.ELISA results showed that PRL content in serum of ES mice was significantly increased compared with WT mice,while PRL content in serum of TLR4-/-mice injected with ES was significantly decreased compared with THAT of ES mice.Western blot results showed that compared with WT mice,pituitary PRL and ERβ/TLR4/p38MAPK pathway proteins in ES mice were significantly increased,while pituitary PRL and ERβ/TLR4/p38MAPK pathway proteins in TLR4-/-mice injected with ES were significantly decreased.（4）Pharmacodynamics of TLR4 inhibitor TAK242 on prolactinoma mice.Weighing results showed that compared with Con group,pituitary weight/body weight of mice in ES group was significantly increased,while compared with ES group,pituitary weight/body weight of mice in ES+Bro group was significantly decreased,and pituitary weight/body weight of mice in ES+TAK242（0.15mg/kg）group was significantly decreased.Pituitary weight/body weight was significantly decreased in ES+TAK242（0.3mg/kg）group.ELISA results showed that compared with Con group,serum PRL content in ES group was significantly increased,but compared with ES group,serum PRL content in ES+Bro group was significantly decreased,and that in ES+TAK242（0.15mg/kg）group was significantly decreased.Serum PRL content of ES+TAK242（0.3mg/kg）group was significantly decreased;Western blot results showed that compared with Con group,pituitary PRL and TLR4/p38MAPK pathway proteins in ES group were significantly increased,while compared with ES group,pituitary PRL and TLR4/p38MAPK pathway proteins in ES+Bro group were significantly decreased.The pituitary PRL and TLR4/p38MAPK pathway proteins were significantly decreased in ES+TAK242（0.15mg/kg）group,and the pituitary PRL and TLR4/p38MAPK pathway proteins were significantly decreased in ES+TAK242（0.3mg/kg）group.（5）TAK242 liposomes were prepared by thin film dispersion method.Taking encapsulation rate as the index,the optimal formulation was selected as follows:TAK242:lecithin:cholesterol=1:10:1,ultrasonic power was 150W,and the size of the liposomes was suitable,the morphology was round under electron microscope,and the encapsulation rate was high.Conclusion:The TLR4/p38MAPK pathway is involved in the pathogenesis of estrogen-induced prolactinoma.While inhibition of TLR4 inhibits secretion and synthesis of pituitary prolactin,suggesting that TLR4 may be a therapeutic target for prolactin tumor. |
