| Background and PurposePrimary aldosteronism(PA)is a disease in which the glomerular zone of the adrenal cortex autonomously secretes excess aldosterone.PA is one of the common causes of secondary hypertension.Aldosterone adenoma occurs in the adrenal cortex accounting for 40-50% of PA.It can lead to hypertension and severe target organ damage.The cause of the disease is not yet clear,which may be related to somatic gene mutations.The WNT/β-catenin signaling pathway exists in numerous tissues and organs of the human body.Abnormal activation of the WNT pathway is closely related to the occurrence and development of numerous tumors,so the WNT pathway is often used as a target for the treatment and research of various tumors.Multiple clinical trials have shown frequent activation of WNT/ β-catenin signals in the adrenal glands of APA patients,indicating WNT/ β-catenin signaling pathway may be an important signaling pathway for the development of adrenal cortical tumors.Secreted frizzledrelated protein 2(s FRP2)is considered to be one of the strongest antagonists of WNT.It is localized on chromosome 4q31 and contains two introns and three exons.s FRPs family has five members: s FRP1,s FRP2,s FRP3,s FRP4 and s FRP5.Among them,s FRP2 may block WNT by competing with curly proteins to bind to WNT ligands.The activation of the β-catenin signaling pathway is widely involved in cell proliferation,apoptosis,and differentiation.Whether s FRP2 is involved in disease progression in APA through inhibition of the WNT/β-catenin signaling pathway has not been reported.In the first part of this thesis,the expression of s FRP2 and the WNT5a/β-catenin signaling pathway in APA adrenal tissues was investigated in human adrenal glands.In the second part of this thesis,we investigated whether s FRP2 is involved in affecting the adrenal cortical cell apoptosis,cell migration and invasion ability,and the expression of molecules related to aldosterone synthesis channels through the classical WNT/β-catenin signaling pathway.In the third part of this paper,we observed changes in body weight,blood pressure,aldosterone secretion,renin activity,and expression of downstream signaling molecules in the WNT/ β-catenin signaling pathway by infusing a lentivirus vector overexpressing s FRP2 and administering a WNT pathway activator into rats.Further exploration the role of s FRP2、WNT/β-catenin pathway in the occurrence and development of APA.Part I Clinical research: The study of the expression of s FRP2,WNT5a/β-catenin pathway in aldosteronism and normal adrenal tissuesObjective: To investigate the expression of human s FRP2 and WNT5a/β-catenin molecule signaling pathway in APA adrenal tissue.Methods: 16 APA and 11 normal adrenal tissues of patients who were hospitalized in the Second Affiliated Hospital of Anhui Medical University from May 2019 to May2022 were collected.a)Organizational structure was abserved between adrenal gland of APA and normal tissue by HE staining.b)The difference in β-catenin expression between the two groups was detected by immunohistochemistry.c)m RNA expression of s FRP2 and WNT5 a between the two groups were detected by RT-PCR.d)Western Blot(WB)was used to detect the expression of s FRP2,WNT5 a,β-catenin,LEF1,TCF,and aldosterone synthetase(CYP11B2)between the two groups.Results: a)HE staining showed that eosinophilic spironolactone bodies were visible in the cytoplasm.b)Immunohistochemistry showed that β-catenin expression in adrenal tissues of APA patients was significantly higher than that in normal adrenal tissues.c)RT-PCR showed that the m RNA of s FRP2 was down-regulated in APA adrenal tissue,while the m RNA of WNT5 a was significantly up-regulated.WB showed that s FRP2 was low in APA adrenal tissue,while WNT pathway proteins WNT5 a,β-catenin,LEF1,TCF and CYP11B2 were high in APA adrenal tissue.Conclusion: The expression of s FRP2 was low in APA adrenal tissue.WNT5a/β-Catenin related signaling pathways were highly expressed in APA.Part II Cell experiment: The study of s FRP2 on the Effect of Cell Phenotype through WNT/β-cateninObjective: Exploring whether s FRP2 regulates WNT/β-catenin pathway affects the cellular phenotype of human adrenal cortical adenocarcinoma cells(NCIH295R).Methods: a)NCI-H295 R cells were divided into two groups: KYA1797K(WNT/β-catenin pathway inhibitor)inhibitor group and control group.Cell apoptosis and the cell cycle were detected by flow cytometry.Cell migration ability was detected by scratch test.Transwell invasion chamber assay was used to monitor cell invasion ability.MTT assay was used to compare cell proliferation between the two groups.The expression of β-catenin m RNA was detected by RT-PCR.The protein expression differences of LEF1,TCF,and CYP11B2 between the two groups were compared by WB.b)NCI-H295 R cells were divided into the sh-s FRP2 group,Lv-s FRP2 group,and control group(blank vector).Cell apoptosis and the cell cycle were detected by flow cytometry.Cell migration ability was detected by scratch test.Transwell invasion chamber assay was used to monitor cell invasion ability.MTT was used to compare the cell proliferation activity among the three groups.RT-PCR was used to detect the m RNA expression of s FRP2 and β-catenin in the three groups.The protein expressions of β-catenin,LEF1,TCF,and CYP11B2 in the three groups were detected by Western blot.c)NCI-H295 R cells were divided into three groups: Control group,Lv-s FRP2 group and Lv-s FRP2+SKL200L group.Cell apoptosis and cell cycle were detected by flow cytometry.Monolayer cell scratch test was used to detect cell migration ability among the three groups.Transwell invasion chamber assay was used to monitor cell invasion ability.MTT was used to compare the cell proliferation activity among the three groups.RT-PCR was used to detect the m RNA expression of s FRP2 and β-catenin in the three groups.The protein expressions of β-catenin,LEF1,TCF and CYP11B2 in the three groups were detected by Western blot.Results: a)Compared with the control group,KYA1797 K group could promote the apoptosis of NCI-H295 R cells,increase the number of cells in G1 phases,significantly inhibit the migration ability of NCI-H295 cells and reduce the invasion ability of NCI-H295 cells.The ability of cell proliferation was also decreased significantly.KYA1797 K inhibited β-catenin expression in NCI-H295 R.Protein expression of WNT/β-catenin signaling pathway LEF1,TCF,and CYP11B2 in the KYA1797 K inhibition group were significantly inhibited.b)Compared with the sh-s FRP2 group,overexpression of s FRP2 significantly promoted the apoptosis of NCI-H295 R cells,increased the number of cells in G1 phases,inhibited the migration and invasion ability of NCI-H295 R cells.The cell proliferation ability was also significantly reduced.The m RNA expression of β-catenin was down-regulated.WB showed that the protein expressions of β-catenin,LEF1,TCF and CYP11B2 in WNT/β-catenin signaling pathway were significantly decreased in the s FRP2 overexpression cell group.c)Addition of WNT/β-catenin signaling pathway agonist reversed the effect of overexpressing s FRP2 in NCI-H295 R cells.Conclusion: KYA1797 K could inhibit WNT/β-catenin signaling pathway and the cell phenotype of NCI-H295 R cells.Overexpression of s FRP2 could inhibit WNT/β-catenin pathway and the cell phenotype of NCI-H295 R cells.However,addition of SKL200 L reversed the effect of overexpressing s FRP2.Part III Animal experiment: The study of s FRP2 in aldosterone-producing adenoma through WNT/β-catenin pathwayObjective: To explore whether s FRP2 affected blood pressure,the aldosterone secretion and proteins of upstream channel by influencing WNT/β-catenin pathway at the animal level.Methods: a)SD rats were divided into two groups: aldosterone tumor model group and control group.The changes in body weight,blood pressure,tissue morphology,aldosterone concentration,renin activity and s FRP2 expression level of the rats were detected.b)After modeling,APA rats were divided into control group,Lv-s FRP2 group,and Lv-s FRP2+SKL2001 group.After 4 weeks,the body weight,aldosterone concentration,renin activity,blood pressure of the three groups of rats were measured;RT-PCR and WB methods were used to detect the levels of m RNA and protein expression levels of β-catenin,LEF1,TCF,and CYP11B2.Results: a)From the weight change curve of rats,it can be seen that the weight of the control group has been increasing over time,while the weight of the aldosterone tumor model group has not significantly increased,and growth and development are hindered;The HE staining results showed that the aldosterone model group had severe inflammation due to the dark red cytoplasm color;The concentration of aldosterone was significantly higher than that of the control group,and the activity of renin was significantly lower than that of the control group;The immunohistochemical results showed that the expression level of s FRP2 in the aldosterone model group was significantly lower than that in the control group.b)Compared to the control group,the body weight of SD rats in the Lv-s FRP2 group recovered significantly,and the body weight of SD rats in the Lv-s FRP2+SKL2001 group decreased significantly compared to the Lv-s FRP2 group.Compared with the control group,the PAC of SD rats in the Lv-s FRP2 group significantly decreased,while the PAC of SD rats in the Lv-s FRP2+SKL2001 group significantly increased compared to the Lv-s FRP2 group.Compared with the control group,the PRA of SD rats in the Lv-s FRP2 group significantly increased,while the PRA of SD rats in the Lv-s FRP2+SKL2001 group significantly decreased compared to the Lv-s FRP2 group.Compared with the control group,the blood pressure of SD rats in the Lv-s FRP2 group significantly decreased,while the blood pressure of SD rats in the Lv-s FRP2+SKL2001 group significantly increased compared to the Lv-s FRP2 group.Compared to the control group,The m RNA and protein expression levels of the WNT/ β-catenin pathway molecule LEF-1,TCF,and CYP11B2 of SD rats in the Lv-s FRP2 group were significantly reduced,but the m RNA and protein expression levels of SD rats in the Lv-s FRP2+SKL2001 group was significantly induced compared to the Lv-s FRP2 group.These all indicate that inhibiting the WNT/β-catenin pathway can weaken the development of aldosterone adenomas in SD rats induced by s FRP2,which is beneficial for disease recovery.Conclusion: Increasing s FRP2 expression could decrease arterial blood pressure and aldosterone secretion in APA rats through WNT/β-catenin pathway. |
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