Study On The Role Of NLRP3 Inflammasome In Prolactinomas And The Therapeutical Effect Of Its Inhibitor MCC950

Background Prolactinomas is a functional pituitary adenoma characterized by excessive prolactin secretion,and the pathogenesis is still unclear.The treatment methods included surgical resection and bromocriptine,but there are some disadvantages such as serious drug resistance.Therefore,it is urgent to explore the pathogenesis of prolactinomas,and develop new therapeutic drugs.Abnormal activation of NLRP3 inflammasome is closely related to many major diseases,such as tumor,chronic kidney disease,joint disease,venous thrombosis and so on.Inflammatory cytokine IL-1β and IL-18 mediated by NLRP3 inflammasome have been confirmed to promote the occurrence of a variety of tumors,such as glioma,nasopharyngeal carcinoma,head and neck squamous cell carcinoma.Objective This study aims to investigate the role and mechanism of NLRP3 inflammasome in pituitary prolactinomas,and the therapeutic effect of small molecule inhibitor MCC950 targeting NLRP3 inflammasome.The study included six parts:1)to investigate the pituitary inflammation and NLRP3 inflammasome expression of prolactinomas animal model;2)to study NLRP3 inflammasome expression and location of prolactinomas human body specimens;3)to study the effect of bromocriptine on pituitary NLRP3 inflammasome expression in prolactinomas rats;4)to study the effect of NLRP3 gene knockout on pituitary gland and prolactin in prolactinomas mice;5)to study the effect of NLRP3 inflammasome activation on prolactin expression of pituitary tumor cells;6)to study the effect of NLRP3 inflammasome inhibitor MCC950 on pituitary gland growth and prolactin expression in prolactinomas rats.Part 1 Pituitary inflammation and NLRP3 inflammasome expression of prolactinomas animal modelPurpose:To study the pituitary inflammation and the expression of NLRP3 inflammasome in prolactinomas model rats.Method:The prolactinomas model rats were established by intraperitoneal injection of estradiol benzoate at a dose of 20 mg/kg.1)PET-CT imaging was used to detect inflammation in rat pituitary tissue;2)Differentially expressed genes and signaling pathways in rat pituitary gland were analysed by microarray;3)Serum prolactin（PRL）protein level was detected by ELISA;4)PRL and NLRP3 inflammasome expression in rat pituitary gland were detected by Western blotting;5)NLRP3 inflammasome expression in rat pituitary gland was analysed by immunofluorescence assay.Results:1)Compared with control group,translocator protein（TSPO）level of pituitary gland was elevated in estradiol-induced prolactinomas rats significantly;2)Compared with control group,the innate immune response gene was significantly upregulated in the pituitary gland of estradiol-induced prolactinomas rats;3)Compared with control group,serum PRL level was increased in estradiol-induced prolactinomas rats significantly;4)Compared with control group,PRL protein and NLRP3 inflammasome expression of pituitary glands were increased in estradiol-induced prolactinomas rats significantly;5)Compared with control group,NLRP3 inflammasome expression of pituitary glands was increased in estradiol-induced prolactinomas rats significantly.Part 2 NLRP3 inflammasome expression and location of prolactinomas human body specimensPurpose:To study the NLRP3 inflammasome expression and location of prolactinomas human body specimens.Method:1)NLRP3 inflammasome expression of prolactinomas human body specimens was analysed by immunohistochemistry;2)NLRP3 inflammasome expression of prolactinomas human body specimens was analysed by immunofluorescence assay.Results:1)Compared with nonfunctioning pituitary specimens,NLRP3 protein expression of prolactinomas human body specimens was increased significantly;2)NLRP3 protein was colocated with ionized calcium binding adapter molecule 1（IBA-1）protein in prolactinomas human body specimens;3)Compared with nonfunctioning pituitary specimens,ASC protein expression of prolactinomas human body specimens was increased significantly.Part 3 Effect of bromocriptine on pituitary NLRP3 inflammasome expression in prolactinomas ratsPurpose:To study the effect of bromocriptine on pituitary NLRP3 inflammasome expression in prolactinomas rats.Method:The prolactinomas model rats were established by intraperitoneal injection of estradiol benzoate at a dose of 20 mg/kg.1)Serum PRL protein level was detected by ELISA;2)PRL and NLRP3 inflammasome expression in rat pituitary gland were detected by Western blotting.Results:1)Compared with control group,the serum PRL level was significantly increased in estradiol-induced prolactinomas rats;compared with model group,the serum PRL levels were both significantly decreased in model rats administrated with bromocriptine（0.9 mg/kg or 1.8 mg/kg）;2)Compared with control group,the pituitary PRL protein expression was significantly increased in estradiol-induced prolactinomas rats;compared with model group,the pituitary PRL protein expression were significantly decreased in model rats administrated with bromocriptine（1.8 mg/kg）.Compared with control group,the pituitary NLRP3 inflammasome protein expression were significantly increased in estradiol-induced prolactinomas rats;compared with model group,the pituitary NLRP3 inflammasome protein expression were significantly decreased in model rats administrated with bromocriptine（1.8 mg/kg）.Part 4 Effect of NLRP3 gene knockout on pituitary gland and prolactin in prolactinomas micePurpose:To study the effect of NLRP3 gene knockout on pituitary gland and prolactin in prolactinomas mice.Method:1)The ratio of pituitary weight to body weight（mg/g）in mice was calculated;2)Serum PRL protein level was detected by ELISA;3)PRL and NLRP3 inflammasome expression in mice pituitary gland were detected by Western blotting;4)NLRP3 inflammasome expression in mice pituitary gland was analysed by immunohistochemistry.Results:1)Compared with wild type（WT）mice,the ratio of pituitary weight to body weight（mg/g）were significantly increased in estradiol-induced or DRD2（-/-）prolactinomas mice;compared with model group,the ratio of pituitary weight to body weight（mg/g）were significantly decreased in NLRP3（-/-）mice induced by estradiol or DRD2 deletion;2)Compared with wild type（WT）mice,the serum PRL levels were significantly increased in estradiol-induced or DRD2（-/-）prolactinomas mice;compared with model group,the serum PRL levels were significantly decreased in NLRP3（-/-）mice induced by estradiol or DRD2 deletion;3)Compared with wild type（WT）mice,the pituitary PRL and NLRP3 proteins were significantly increased in estradiol-induced or DRD2（-/-）prolactinomas mice;compared with model group,the pituitary PRL and NLRP3 proteins were significantly decreased in NLRP3（-/-）mice induced by estradiol or DRD2 deletion;4)Compared with wild type（WT）mice,the pituitary NLRP3 protein expression were significantly increased in estradiol-induced or DRD2（-/-）prolactinomas mice;compared with model group,the pituitary NLRP3 proteins were not expressed in NLRP3（-/-）mice and NLRP3（-/-）mice induced by estradiol or DRD2 deletion.Part 5 Effect of NLRP3 inflammasome activation on prolactin expression of pituitary tumor cellsPurpose:To study the effect of NLRP3 inflammasome activation on prolactin expression of pituitary tumor cells.Method:1)PRL and NLRP3 inflammasome expression were detected in GH3 cells after active pro-inflammatory agents were added;2)PRL and NLRP3 inflammasome expression were detected in GH3 cells after active pro-inflammatory agents and NLRP3 inflammasome inhibitor MCC950 were added.Results:1)Compared with control group,PRL,NLRP3,Caspase-1,Pro-IL-1β,and IL-1β proteins expression of cell lysate were significantly increased in LPS-stimulared group;the same results were also detected in GH3 cells stimulared by active pro-inflammatory agents such as LPS+MSU,LPS+ATP,or LPS+Ni;2)Compared with control group,PRL,NLRP3,Caspase-1,Pro-IL-1β,and IL-1β proteins expression of cell lysate were significantly increased in LPS and Ni-stimulared group,which were reversed by 10 μM concentration of MCC950.Part 6 Effect of NLRP3 inflammasome inhibitor MCC950 on pituitary gland growth and prolactin expression in prolactinomas ratsPurpose:To study the effect of NLRP3 inflammasome inhibitor MCC950 on pituitary gland growth and prolactin expression in prolactinomas rats.Method:The prolactinomas model rats were established by intraperitoneal injection of estradiol benzoate at a dose of 20 mg/kg.1)The ratio of pituitary weight to body weight（mg/g）in rats was calculated;2)Serum PRL protein level was detected by ELISA;3)PRL and NLRP3 inflammasome expression in rat pituitary gland were detected by Western blotting.Results:1)Compared with control group,the ratio of pituitary weight to body weight（mg/g）was significantly increased in estradiol-induced prolactinomas rats;compared with model group,the ratio of pituitary weight to body weight（mg/g）was significantly decreased in model rats administrated with MCC950（10 mg/kg or 20 mg/kg）;2)Compared with control group,the serum PRL level was significantly increased in estradiol-induced prolactinomas rats;compared with model group,the serum PRL levels were both significantly decreased in model rats administrated with MCC950（10 mg/kg or 20 mg/kg）;3)Compared with control group,the pituitary PRL protein expression was significantly increased in estradiol-induced prolactinomas rats;compared with model group,the pituitary PRL protein expression were significantly decreased in model rats administrated with MCC950（20 mg/kg）.Compared with control group,the pituitary NLRP3 inflammasome protein expression were significantly increased in estradiol-induced prolactinomas rats;compared with model group,the pituitary NLRP3 inflammasome protein expression were significantly decreased in model rats administrated with MCC950（20 mg/kg）.Conclusion:The NLRP3 inflammasome activation in pituitary gland promotes the development of prolactinomas was found firstly,and NLRP3 inflammasome inhibitor MCC950 shows effective anti-prolactinomas effect.