| Background and Objective:Benign prostatic hyperplasia(BPH)is the most common disorder in the elderly.The research results of most domestic and foreign scholars show that chronic prostatic inflammation is closely related to BPH in epidemiology,which means that chronic prostatic inflammation plays a very important role in the occurrence and development of BPH,and more prostatic inflammation is histological inflammation.Histological inflammation is far more than chronic prostatitis with clinical manifestations.Prostate development and growth are regulated by androgens and growth factors,among which growth factors secreted by prostate epithelial cells and mesenchymal cells play a key role.The expression level of these factors is low in normal prostate tissue,and the expression level is increased in benign hyperplasia prostate tissue,which will promote the further growth of the prostate.Prostate epithelial and mesenchymal cells can express fibroblast growth factor(FGF)receptors,and can also synthesize and secrete FGF.Under normal circumstances,FGF maintains the stable state of prostatic interstitial cells,and acts as the autocrine of interstitial cells to induce the remodeling of prostate tissue.Transforming growth factor-β(TGF-β)is a multifunctional inflammatory cytokine involved in the regulation of the biological processes of prostate cell proliferation,apoptosis,migration and differentiation.In the FGF family and TGF-β family,FGF2 and TGF-β1 are the most representative growth factors in the prostate.In recent years,domestic and foreign scholars have made certain explorations on the role of FGF2 and TGF-β1 in the occurrence and development of BPH in chronic prostatic inflammation.At present,researchers have found that interleukin 6(IL6)and interleukin 8(IL8)are involved in the proliferation of prostate tissue,but there is no clear report on whether IL6 and IL8 are related to changes in FGF2 and TGF-β1 levels.Therefore,in the process of prostate tissue hyperplasia,whether IL6 and IL8 are involved in regulating FGF2 and TGF-β1 has become the core issue that we intend to solve in this research.Methods:Part Ⅰ: The clinical data and BPH specimen tissues of 50 patients undergoing transurethral resection of the prostate(TURP)were collected.All patients underwent digital rectal examination and color Doppler ultrasonography of the prostate before the operation and there was no obvious abnormality.The pathological examination of the prostate tissue in all patients after the operation was benign prostatic hyperplasia.According to the preoperative white blood cell(WBC)count in the expressed prostatic secretion(EPS),the patients were divided into groups and combined with the clinical data of the patients,the relationship between BPH tissue inflammation and clinical indicators was explored.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of IL6,IL8,FGF2 and TGF-β1 in EPS of all BPH patients,and analyze their correlation with various clinical indicators.Hematoxylin-eosin(HE)staining and immunohistochemistry(IHC)were used to detect the degree of inflammation and the expression of FGF2 and TGF-β1 in prostate tissue in all patients.The purpose is to explore the expression levels of FGF2 and TGF-β1 in chronic tissue inflammation and the relationship with BPH through clinical data.Part Ⅱ: By further culture of human benign prostatic hyperplasia epithelial cell line(BPH-1),and the expression of FGF2 and TGF-β1 and cell status in BPH-1 cells were detected by real-time polymerase chain reaction(Real-time PCR),Western blot(WB),immunofluorescence and 5-ethynyl-2’-deoxyuridine(Ed U)under the stimulation of exogenous lipopolysaccharide(LPS),TGF-β and the TGF-βinhibitors.Part Ⅲ: It was further verified on the rat model that the intraprostatic injection of LPS in the rat caused inflammation of the rat prostate,which in turn induced the establishment of the rat BPH model.At the same time,the rat BPH model was given TGF-β inhibitor(SB-431542)and grouped.The IHC method was used to detect the expression of FGF2 and TGF-β1 in the prostate tissue of each group of rats.ELISA was used to detect the concentration of IL6 and IL8 in the serum and prostate tissue of each group of rats.Real-time PCR and WB were used to detect the m RNA and protein expression of FGF2 and TGF-β1 in the prostate tissues of rats in each group to explore the effect of increased FGF2 and TGF-β1 expression on BPH caused by inflammation of the prostate tissue.Results:Part Ⅰ:1.In this study,clinical data and prostate tissue specimens of 50 BPH patients were collected.According to the WBC count of EPS samples before the operation,there were 31 cases of BPH tissue with inflammation;compared to BPH,prostate volume,IPSS score,TPSA were statistically significant(P<0.05).2.In the clinical data of 31 BPH patients with inflammation,the relevant clinical indicators were: age and cholesterol(R=-0.357,P=0.049),c-reactive protein(R=0.493,P=0.005);prostate volume and IPSS score(R=0.553,P=0.001),c-reactive protein(R=0.474,P=0.007);IPSS score and BMI(R=0.387,P=0.032),c-reactive protein(R=0.451,P=0.011);BMI and fasting blood glucose(R=0.405,P=0.024),high-density lipoprotein(R=-0.467,P=0.008);cholesterol and low-density lipoprotein(R=0.731,P<0.001).3.Through ELISA detection of the indicators in EPS in 50 BPH patients and analysis of their correlation with various clinical indicators,it was found that the prostate volume(Z=-4.850,P<0.001),IPSS score(t=-5.630,P<0.001),TPSA(Z=-2.738,P=0.006),IL8(t=-26.100,P<0.001),FGF2(t=-20.080,P<0.001)and TGF-β1(t=-21.610,P<0.001)are greater than patients with low IL6 concentration.The prostate volume of patients with high IL8 concentration(Z=-4.601,P<0.001),IPSS score(t=-5.510,P<0.001),TPSA(Z=-2.618,P=0.009),IL6(t=-13.610,P<0.001),FGF2(t=-14.860,P<0.001)and TGF-β1(t=-18.780,P<0.001)are all greater than those with low IL8 concentration.The prostate volume of patients with high FGF2concentration(Z=-4.773,P<0.001),IPSS score(t=-5.400,P<0.001),TPSA(Z=-2.355,P=0.019),IL6(t=-18.830,P<0.001),IL8(t=-21.450,P<0.001)and TGF-β1(t=-18.000,P<0.001)are all greater than those with low FGF2 concentration.The prostate volume of patients with high TGF-β1 concentration(Z=-4.571,P<0.0 0 1),IPSS score(t=-5.100,P<0.001),TPSA(Z=-2.506,P=0.012),IL6(t=-13.350,P<0.001),IL8(t=-19.950,P<0.001)and FGF2(t=-14.220,P<0.001)are greater than those with low TGF-β1 concentration.The above differences are statistically significant significance.4.The prostate tissues of 50 BPH patients were detected by HE staining,and it was found that 96%(48/50)of BPH tissues had inflammation of different degrees.IHC detected all BPH patients and found that regardless of whether the hyperplastic gland tissues were complicated with inflammation,prostate epithelial cells and mesenchymal cells have the expression of TGF-β1 and FGF2,and the degree of inflammation is positively correlated with the expression of TGF-β1 and FGF2.Part Ⅱ:1.Under LPS stimulation,the LPS group compared with the control group:(1)Real-time PCR detected the expression of FGF2 and TGF-β1 m RNA in BPH-1 cells at 2 h,6 h,12 h,24 h and 48 h,respectively(0.983±0.011 vs 1.008±0.011,P=0.070),(1.169±0.024 vs 1.000±0.046,P=0.010),(1.112±0.079 vs 0.958±0.058,P=0.079),(1.773±0.033 vs 0.936±0.016,P<0.001),(1.280±0.009 vs 0.968±0.042,P= 0.001),(1.924±0.071 vs 1.020±0.022,P<0.001),(1.468±0.089 vs 1.173±0.102,P=0.037),(2.381±0.067 vs 1.133±0.050,P<0.001),(1.568±0.068 vs 1.075±0.007,P=0.001),(2.429±0.067 vs 0.983±0.008,P<0.001).The expressions of FGF2 and TGF-β1 m RNA in the LPS group increased significantly with the increase of time,and the difference was statistically significant.(2)WB detected FGF2 and TGF-β1 protein expression in BPH-1 cells at 6 h,12 h,24 h,48 h,respectively(1.033±0.032 vs 0.988±0.085,P=0.520),(1.220±0.063 vs1.000±0.072,P=0.031),(1.084 ±0.008 vs 0.970±0.038,P=0.015),(1.245±0.040 vs1.000±0.116,P=0.048),(1.143±0.037 vs 0.985±0.057,P=0.031),(1.550±0.054 vs0.999±0.0 30,P<0.001),(1.295±0.047 vs 1.001±0.011,P=0.001),(1.565±0.038 vs1.084±0.171,P=0.018).The expressions of FGF2 and TGF-β1 protein in the LPS group increased significantly with the increase of time,and the difference was statistically significant.2.Under TGF-β stimulation,the TGF-β group compared with the control group:(1)Real-time PCR detected the expression of FGF2 and TGF-β1 m RNA expression in BPH-1 cells at 2 h,6 h,12 h,24 h,and 48 h,respectively(1.054±0.035 vs 1.008±0.011,P=0.155),(1.125±0.059 vs 1.000±0.046,P=0.077),(1.135±0.074 vs0.958±0.058,P=0.046),(1.141±0.027 vs 0.936±0.016,P=0.001),(1.345±0.039 vs0.968±0.042,P=0.001),(1.376±0.025 vs 1.020±0.022,P<0.001),(1.521±0.048 vs1.173±0.102,P=0.012),(1.369±0.025 vs 1.133±0.050,P=0.004),(1.652±0.036 vs1.075±0.007,P<0.001),(1.727±0.054 vs 0.983±0.008,P<0.001).The expressions of FGF2 and TGF-β1 m RNA in the TGF-β group increased significantly with the increase of time,and the difference was statistically significant.(2)WB detected FGF2 and TGF-β1 protein expression in BPH-1 cells at 6 h,12 h,24 h,48 h,respectively(1.082±0.026 vs 0.988±0.085,P=0.209),(1.223±0.030 vs1.000±0.072,P=0.015),(1.174±0.049 vs 0.970±0.038,P=0.007),(1.292±0.038 vs1.000 ± 0.116,P=0.028),(1.279±0.060 vs 0.985±0.057,P=0.002),(1.293±0.062 vs0.999± 0.030,P=0.004),(1.389±0.051 vs 1.001±0.011,P<0.001),(1.493±0.052 vs1.084±0.171,P=0.032).The expressions of FGF2 and TGF-β1 protein in the TGF-βgroup increased significantly with the increase of time,and the difference was statistically significant.3.Under the inhibition of SB-431542,the SB-431542 group was compared with the control group:(1)Real-time PCR detected the expression of FGF2 and TGF-β1 m RNA expression in BPH-1 cells at 2 h,6 h,12 h,24 h,and 48 h respectively(1.041±0.007 vs 1.008±0.011,P=0.021),(1.000±0.035 vs 1.000±0.046,P=0.998),(0.747±0.055 vs0.958±0.058,P=0.014),(1.063±0.073 vs 0.936±0.016,P=0.074),(0.585±0.026 vs0.968±0.042,P<0.001),(0.804±0.008 vs 1.020±0.022,P<0.001),(0.456±0.025 vs1.173±0.102,P<0.001),(0.774±0.016 vs 1.133±0.050,P=0.001),(0.434±0.029 vs1.075±0.007,P<0.001),(0.672±0.008 vs 0.983±0.008,P<0.001).The m RNA expression levels of FGF2 and TGF-β1 in the SB-431542 group decreased significantly with the increase of time,and the difference was statistically significant.(2)WB detected FGF2 and TGF-β1 protein expression in BPH-1 cells at 6 h,12 h,24 h,and 48 h respectively(0.842±0.084 vs 0.988±0.085,P=0.158),(0.945±0.013 vs 1.000±0.072,P=0.343),(0.731±0.089 vs 0.970±0.038,P=0.025),(0.883±0.065 vs1.000±0.116,P=0.277),(0.673±0.015 vs 0.985±0.057,P=0.002),(0.800±0.040 vs0.999±0.030,P=0.005),(0.568±0.070 vs 1.001±0.011,P=0.001),(0.564±0.030 vs1.084±0.171,P=0.013).The protein expression levels of FGF2 and TGF-β1 in the SB-431542 group decreased significantly with the increase of time,and the difference was statistically significant.4.Through immunofluorescence detection,compared with the control group,the expression of FGF2 and TGF-β1 in the LPS and TGF-β group was significantly increased,while the expression of FGF2 and TGF-β1 in the SB-431542 group was significantly decreased.5.The Edu method detected BPH-1 cells and found that the proliferation ability of the cells in the LPS group and TGF-β group increased significantly with the increase of time,while the proliferation ability of the cells in the SB-431542 group gradually decreased with the increase of time.Part Ⅲ:1.The rat prostate hyperplasia model was successfully established by injecting 100 μg/kg LPS into the rat prostate through the urethra.2.IHC detected the prostate tissue of each group of rats.FGF2 and TGF-β1 were mainly expressed in epithelial(including glandular epithelial)cells,fibroblasts in the mesenchyme,inflammatory cells,and endothelial cells of blood vessels,especially glandular epithelial cells.Compared with the Control group,the expression of FGF2 and TGF-β1 in the prostate tissue of the Model group were significantly increased.Compared with the Model group,the expression of FGF2 and TGF-β1 in the prostate tissue of the SB-431542 group was significantly reduced.3.When the Model group is compared with the Control group:(1)ELISA detected the concentrations of IL6(30.79±2.62 pg/ml vs 7.83±1.86pg/ml,P<0.001),IL8(20.07±1.07 pg/ml vs 4.06±0.55 pg/ml,P<0.001)in rat serum and the concentrations of IL6(184.71±3.84 pg/ml vs 87.06±3.66 pg/ml,P<0.001),IL8(113.63±2.70 pg/ml vs 26.81±1.63 pg/ml,P<0.001)in rat prostate tissue were significantly increased,and the difference is statistically significant.(2)Real-time PCR detection showed that the m RNA expression levels of FGF2(2.119±0.977 vs 1.407±0.693,P=0.115)and TGF-β1(1.527±0.942 vs 0.517±0.277,P=0.011)were increased in the prostate tissues of rats.However,only the expression difference of TGF-β1 m RNA was statistically significant.(3)WB detection showed that the protein expression levels of FGF2(1.858±0.042 vs 1.000±0.052,P<0.001)and TGF-β1(2.235±0.049 vs 0.993±0.020,P<0.001)were significantly increased in the prostate tissue of rats,and the difference was statistically significant.4.When the SB-431542 group is compared with the Control group:(1)ELISA detected the concentrations of IL6(15.95±1.60 pg/ml vs 7.83±1.86pg/ml,P<0.001),IL8(13.93±0.98 pg/ml vs 4.06±0.55 pg/ml,P<0.001)in rat serum and the concentrations of IL6(134.94±3.79 pg/ml vs 87.06±3.66 pg/ml,P<0.001),IL8(104.06±3.76 pg/ml vs 26.81±1.63 pg/ml,P<0.001)in rat prostate tissue were significantly increased,and the difference was statistically significant.(2)Real-time PCR detection showed that the expression of FGF2(0.800±0.477 vs 1.407±0.693,P=0.061)and TGF-β1 m RNA(0.363±0.168 vs 0.517±0.277,P=0.199)were decreased in the prostate tissues of rats,but there was no significant difference between the two groups.(3)WB detection showed that the protein expression levels of FGF2(1.161±0.004 vs 1.000±0.052,P=0.006),TGF-β1(1.557±0.060 vs 0.993±0.020,P<0.001)were significantly increased in the prostate tissue of rats,and the difference was statistically significant.5.When the SB-431542 group is compared with the Model group:(1)ELISA detected the concentrations of IL6(15.95±1.60 pg/ml vs 30.79±2.62pg/ml,P<0.001)和 IL8(13.93±0.98 pg/ml vs 20.07±1.07 pg/ml,P<0.001)in rat serum and the concentrations of IL6(134.94±3.79 pg/ml vs 184.71±3.84 pg/ml,P<0.001)、IL8(104.06±3.76 pg/ml vs 113.63±2.70 pg/ml,P<0.001)in rat prostate tissue were significantly decreased,and the difference was statistically significant.(2)Real-time PCR detection showed that the expression expression of FGF2(0.800±0.477 vs 2.119±0.977,P=0.004)and TGF-β1 m RNA(0.363±0.168 vs1.527±0.942,P=0.002)were significantly decreased in the prostate tissues of rats,and the difference was statistically significant.(3)WB detection showed that the protein expression levels of FGF2(1.616±0.004 vs 1.858±0.042,P<0.001),TGF-β1(1.557±0.060 vs 2.235±0.049,P<0.001)were significantly decreased in the prostate tissue of rats,and the difference was statistically significant.Conclusion:1.The levels of inflammatory cytokines in patients with BPH combined with inflammation are significantly increased.BPH combined with inflammation is related to metabolic disease indicators(BMI,fasting blood glucose,cholesterol,high-density lipoprotein,and low-density lipoprotein).FGF2 and TGF-β1 are expressed in hyperplastic prostate tissue,which is positively correlated with the degree of tissue inflammation.Histological inflammation plays an important role in prostate hyperplasia.2.LPS and TGF-β can increase the expression of FGF2 and TGF-β1 and enhance cell proliferation in BPH-1 cells in vitro experiments.Inflammation caused by bacterial infection and other factors and prostate cells themselves may cause BPH through the TGF-β1-FGF2 signaling pathway.3.In vivo experiments,LPS-induced inflammation can cause prostate hyperplasia in rats.Under the stimulation of LPS or inhibition of TGF-β inhibitors,the expression levels of FGF2 and TGF-β1 have changed.Exogenous infection may promote the occurrence and development of BPH through the TGF-β1-FGF2 pathway.In conclusion,tissue inflammation is closely related to BPH,and FGF2 and TGF-β1 play an important role in the process of proliferation. |
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