| Benign prostatic hyperplasia(BPH)is a common disease in middle and aged male. The incidence of the disease is increasing along with coming of long-life society in our country. Up to now, the causes and pathogenesis of the disease is not clear. There have been no breakthroughs on its treatment, especially on pharmacotherapy. The present study was undertaken to further explore the pathogenesis of the disease with respect to expression of estrogen and androgen receptors, related growth factors, apoptosis and proliferation as well as the expression of their related genes at protein level, and ultrastructure of the prostate from patients with BPH. Based on the works, therapeutic mechanisms of estrogen at high dose on BPH were further studied. Finally, according to understanding of prostatic diseases in traditional Chinese medical science, we composed a prescription of traditional Chinese medicine herbs I(TCMH), and studied the therapeutic effects and its mechanisms of the prescription on the experimental prostatic hyperplasia induced by implanting of urogenital sinus in mice. The results are as follows:1. Studies on the pathogenesis of BPHThe following items were compared among transitionalzone(TZ), central zone(CZ) and peripheral zone(PZ) of the prostate from BPH patients: (1) expression of estrogen receptor (ER)and androgen receptor (AR) ; (2) proliferation and apoptosis; (3)expression of bcl-2, bax and c-myc ; (4)expression of epidermal growth factor (EGF), EGF- receptor(EGF-R), basic fibroblast growth factor (bFGF) and transforming growth factor-β 1(TGF-β 1) (5)ultrastructure.The specimens were obtained from 33 patients (aged from 58-83, with mean age 66.4 + 5.3) undergoing prostatectomy for benign prostatic obstruction. The prostates excised were divided into TZ, CZ and PZ according to McNeal method. All the items were quantitatively analysed with flow cytometry(FCM) except for the ultrastructure, which was observed under transmission electron microscope. The expression of above mentioned receptors, genes and growth factors were presented with labeled rate(percentage, %) and expression level (presented by fluorescence channel numbers collected as linear data). Rates of apoptosis and proliferation were expressed by percentage(%) of apoptosis and proliferation. All data were expressed with median for statistics treatment.1. 1 Expression of ER and AR The labeled rate and expression level of ER in the TZ were 26. 86% and 73. 00 respectively, which were significantly higher than those in the CZ(19. 50%,53.50) and PZ(12. 85%, 53. 40) cells of the prostate with BPH(p<0. 01). The AR were highest in the PZ(19.50%, 64. 00) among the three zones and significantly higher than those in the TZ(16.20%, 51.00) (p<0. 01);but there was no significant difference than those in CZ(17. 20%, 60. 00) (p>0. 05).1.2 Rates of proliferation and apoptosis The cell proliferative rate in the TZ(19. 50%) was significantly higherthan that in the CZ(14.00%) and PZ(13.80%) (p<0. 05). No difference in the rate of apoptosis was detected among the three zones.1.3 Expressions of bcl-2, bax and c-myc The labeled rate (25.40%) and expression level (73.00) of bcl-2 in the TZ were significantly higher than those in the PZ (17. 50%, 54. 00)and CZ(17. 70%, 44. 96) (p<0. 01). No difference in either labeled rate and expression level was found for bax. The expression level of c-myc in TZ(52.00) was higher than that in the PZ(44. 96)(p<0. 05), though labeled rate had no difference in the three zones.1. 4 Expression of EGF, EGF-R, bFGF and TGF-β 1 The labeled rate (24. 10%) and expression level (76. 99) of EGF in the TZ were significantly higher than those in the PZ(16. 50%,49.00) and CZ(12. 90%, 50. 99) (p<0. 01). EGF-R showed the same pattern of change with EGF. The labeled rates and expression levels of bFGF in the TZ(22. 50%,68.98) were significantly higher than those in the PZ (16. 40%, 44. 96) and CZ(18. 80%, 44. 96) (p<0. 01) , The labeled rates and expression levels of TGF- β 1 in the TZ(22.60%, 61.98) were significantly higher than those in PZ(15. 20%, 46. 00) and CZ(15. 80%, 50. 00) (p<0. 01)1.5 Ultrastructure Clear ultrastructural differences could be seen among the three zones in BPH prostate. The cells in the TZ showed more vigorously proliferative changes than those in the other two zones. Cytoplasmic membrance merges between lymphocytes and mast cells were found in the TZ.The results revealed first the high expression of ER, bcl-2, c-myc, EGF, EGF-R, bFGF and TGF- β 1, and the vigorously proliferative specificity in ultrastructure in the TZ as compared with the CZ and PZ of the prostate from patients withBPH. These changes may be responsible for inducing over-proliferation of prostatic cells and resulting in BPH.2. Therapeutic effects of high dose of estrogen on BPH and its mechanismsWe observed as a first step effects of high dose of estrogen on size and morphology of the hyperplastic prostate to difine the therapeutic effect of estrogen. Based on this, expressions of ER, AR, bcl-2, c-myc, EGF, EGF-R, bFGF and TGF-0 1, and changes in proliferation and apoptosis rates of prostatic cells under the effects of high dose of estrogen were determined to explore mechanisms underlying the therapeutic effect.Sixty BPH patients were divided into control and treated groups. Each group consisted of 30 patients. All the patients were undergone transbladder prostatectomy. Patients in the treated group were administrated oraly with estrogen (9 mg/day) for 10-14 days (mean 12. 5 days) prior to the operation. Patients in the control group had no administration of the estrogen and were directly undergone the operation. The size of the prostates was measured with ultrasonography transabdominally. The prostates excised were divided into TZ, CZ and PZ according to McNeal method. The morphologic changes were observed after HE staining and the other items were determined by FCM. Collection and presentation of the data were the same as part 1, except size of the prostate, which was presented as mean ±SD in this part.2. 1 Change in size of the prostate The mean size of the prostate was 130.21 + 10. 15 cm3 and 137.52 + 15.75 cm3 in control and treated group before the administration of estrogenrespectively. It was significantly decreased to 116. 05 + 12. 42 cm3 after the estrogen treatment(p<0. 05).2.2 Morphologic changes After estrogen treatment, the prostates in BPH group showed changes suggesting tendency of atrophy of the glands in pathological morphology. The lining epithelia of the glands in BPH were shorten and became short column or cuboidal in shape with deeper nuclear staining. Plicate or papillary structures of glandular epithelia were decreased in number. Retention of prostatic secretion and exfoliated epithelial cells could be seen in a few lumina of glands.2.3 Changes in expression of ER and AR Changes were detected mainly in TZ, represented by significant decreases in labeled rate and expression level of ER(from 26.86% and 73.00 to 18.10% and 46.03 respectively, p<0. 01) and increases in the labeled rate of AR(from 16.20% to 20.55%, p<0. 01) in treated group as compared with control group.2.4 Changes in proliferation and apoptosis Apoptosis rates of the cells in the'TZ (13. 55%), PZ(4. 80%) and CZ(6. 45%) in treated group were increased significantly than that in control group(2. 10%, 2. 40% and 2. 00% respectively, p<0. 01). NO change in rate of proliferation in the TZ was found although it was increased in CZ and PZ in the treated group than that in the control group.2. 5 Changes in expression of bcl-2, bax and c-myc The labeled rate and expression level of bcl-2 in the TZ were significantly decreased from 25.40% and 73.00 to 13.35% and 44. 46 respectively under the effect of estrogen(p<0. 01), while the expression level of bax increased from 48.00 to 56. 97(p<0. 01). In the CZ, the labeled rate of c-myc decreasedfrom 17.80% to 12.20% (p<0. 01). In the PZ, the labeled rate of bcl-2 decreased from 17.50% to 12.80 (p<0.01), the expression level of bcl-2, bax and c-myc decreased significantly from 54. 01, 44. 96 and 44. 96 to 44. 46, 42. 00 and 42. 00 (p<0. 01).2.6 Changes in expression of EGF, EGF-R, bFGF and TGF-β 1 Changes mainly occurred in the TZ. The labeled rate and expression level of EGF in the TZ were significantly decreased from 24. 10% and 76.99 to 14.90% and 47.49 respectively under the effect of estrogen(p<0.01). EGF-R showed the same pattern of changes with EGF. Changes of bFGF and TGF-3 1 represented as a decrease in the expression level under the effect of estrogen (from 68.98 and 61.98 to 59.49 and 56.00, p<0. 01). In addition, the expression level of EGF decreased significantly from 50.99 to 43. 96(p<0. 01) in CZ.The results indicated that the therapeutic effects of high dose of estrogen related with its inhibitory effect on expressions of ER, bcl-2, c-myc, EGF, EGF-R, bFGF and TGF-β 1 and promoting effects on expression of bax and apoptosis in the prostate especially in the TZ. The results further verified the important role of these factors in the pathogenesis of BPH.3. Therapeutic effects and mechanisms of traditional Chinese medicine herbs I on experimental prostatic hyperplasia induced by implanting of the urogenital sinus in miceAccording to understanding of prostatic diseases in Chinese traditional medical science and the results in above studies, we composed a prescription of traditional Chinese medicine herbs I (TCMH), and studied therapeutic effects of theprescription on experimental prostatic hyperplasia induced by implanting of the urogenital sinus and its mechanisms in mice.One hundred twenty Kunming mice(weighting 30-40 g)were divided randomly into 5 groups: (1)control group ; (2)hyperplastic group, hyperplasia of the prostate was induced by implanting of the urogenital sinus; (3)TCMH group, the effects of the TCMH on the hyperplasia were examined; (4) normal saline(NS) group, which served as negative control group; (5)finasteride group(positive control), the effects of finasteride on the hyperplasia were compared with the TCMH. Items observed were weight and morphology of the prostate, expression of ER and AR, rates of apoptosis and proliferation, expression of bcl-2, bax and c-myc and expression of bFGF and TGF-β 1. Collection and presentation of the data were the same as part 1, except weight of the prostate, which was presented as mean + SD, and expression level which was presented by fluorescence channel numbers collected as logarithmic data.3. 1 Change in weight of the prostate The prostates in hyperplastic group(149. 3 + 8. 46 mg) were heavier than those in control group(64. 20 ± 6.02 mg)(p<0.01). No significant difference was detected between the hyperplastic and NS (146.10 + 10.47 mg) groups (p>0. 05). The weight prostate in finasteride group(104. 63 + 19. 60 mg) was lighter than that in NS group (p<0. 01). The weight in the TCMH group (85. 60 + 17. 97 mg) was decreased compared not only with the NS but also with the finasteride group(p<0. 01).3. 2 Morphologic changes Macroscopically, the prostates in BPH group were enlarged and were two times as those of the normal prostates in size. Inflammatory edema in prostate was obvious, thus the tissue was fragile. The enlarged prostateseverely adhered to adjacent bladder and seminal vesicle. Similar pathological changes were found in the prostates of negative controls. The enlarged prostates in positive control group were smaller than those in BPH group and larger than those of normal controls. Adhesion to adjacent organs, obvious edema could also be found in this group and the prostates were fragile. While the prostates in the TCMH group were about the same as normal controls in size and no inflammation, edema and adhesion could be seen in prostates. The prostates in this group were relatively firm and elastic. Microscopically, the glandular epithelia showed significant hyperplastic changes and the hyperplastic epithelia formed dense plicate and papillary structures filling the lumina of the prostate glands in BPH and negative control groups. The plicate and papillary structures of epithelia were decreased in number and degenerative and necrostic changes, such as, acidophilic change of cytoplasma, deep nuclear staining, pyknosis, karyorrhexis and exfolication of epithelial cells could be seen in finasteride group. The pathological features of the prostates in the TCMH group were similar to those in finasteride, showing increased lumen secretion and fewer plicate and papillary structures of epithelia as well as a few degenerative, necrotic or exfoliating epithelial cells.3. 3 Changes in the ultrastructure The acinous lumens of the prostate were regular in the normal mouse. There were little connective tissues among the acini. The epithelium cells were column, the nuclei were round and the nucleolus were bigger. There were amount of euchromatin, but seldom of heterochromatin.There were more connective tissues among the acini of the hyperplastic prostate in which the urogenital sinus was implanted to.induce BPH, and there were also masses of smooth muscle cells . There were more conical fundus cells in the acinous epithelium close to the fundus membrane.There appeared masses of macrophage among the acini of the prostate in the finasteride group and there were a great deal of secondary lysosome inside the prostatic cells.The prostatic epithelium cells of the mouse in TCMH group became shorter and changed into short columnal epithelium and cubic epithelium even into flat epithelium. The microvillus in the dissociative surface of cells dismissed and the secretory vacuole decreased significantly. There appeared masses of dissociative ribosome in the cytoplasm.3.4 Changes in expression of ER and AR The labeled rate (18. 90±0. 83) and expression level (4. 91+0. 83) of ER in hyperplastic group were higher than those in the control group (13.03 + 0.94 and 3.87 + 0.25), but those in AR were lower(p<0. 05). The TCMH made the labeled rates of ER(16. 15 + 5.18) and AR(13. 64 + 2. 96) decreased related with hyperplastic and NS groups (p<0.05).3. 5 Changes in rates of apoptosis and proliferation Rates of apoptosis and proliferation in hyperplastic group(3.66 + 0. 73 and 26. 05 + 3. 02) were increased compared with the control (2.14 + 0.33 and 17. 94 + 4. 26) (p<0. 01), but the increase in rate of proliferation was more obvious. The TCMH significantly increased the rate of apoptosis (6. 67 + 1. 91) (p<0.01), but no significant effect on the proliferative rate was found(p>0.05).3.6 Changes in expression of bcl-2, bax and c-mycImmuno-histochemistry staining showed that the expression of bcl-2 in control was weakly positive and that the positivity was increased significantly in hyperplastic group compared with control. The increased expression could be attenuated by treatment with the TCMH. The similar effect to the TCMH was found in finasteride group, but the effect of the TCMH was more pronouced. Changes in bax was contrary to that in bcl-2, represented by a distinct decreased in hyperplastic group related with the control. The TCMH and finasteride presented an similar effect in elevating expression of bax. The expression of c-myc in control was weakly positive and increased significantly in hyperplastic group as compared with control. The increased expression was attenuated significantly by treatment with the TCMH. Finasteride had similar effect to the TCMH but the effect was weaker than the TCMH. Analysis of FCM showed similar trend to that of immunohisto- chemistry observation, represented by significant increases in the labeled rates and expression levels of bcl-2(18. 13±7. 97 and 5. 87±0. 92) and c-myc(15. 95 ±4.34 and 4.46 + 0.70) in hyperplastic group as compared with control group (11. 13 + 1. 06 and 3. 16 + 0. 24 for ER) and (12.36 + 2.97 and 2.89+0.20 for AR)(p<0.01). After treatment with the TCMH, the labeled rate and expression level of bcl-2 (17.13 + 6.86 and 3.81 + 0.75) were decreased(p<0. 01), while that of bax (17. 09 + 6. 17 and 4. 20 + 1.34) were increased significantly (p<0.05) related with hyperplastic and NS groups(p<0.05). Finasteride showed a similar effect on those to the TCMH.3. 7 Changes in expression of bFGF and TGF- β 1 Immunohisto- chemistry analysis showed that the expression of...
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