The Biological Evaluation And Mechanism Study Of Piperazine α₁ Subtype Receptor Blockers Against Benign Prostatic Hyperplasia

Research background:Benign prostatic hyperplasia（BPH）is a common male urinary disease that mostly occurs in middle-aged and elderly people,which incidence increases with age.Prevalence increases to 80%among men 50 to 80 years of age,even more than90%among men after 80 years of age.When prostate obstruction becomes worse,thereby leading to lower urinary tract symptoms（LUTS）.These symptoms can be further classified as obstructive urination or bladder storage disorders.Obstructive urination symptoms include urinary hesitancy,delay in initiating,intermittent incontinence,involuntary interruption of urinary flow,weak urination,incomplete emptying,incomplete terminal urine.Storage disorders,including frequent urination,nocturia,urinary incontinence,bladder pain,or dysuria,which seriously affects patients’quality of life.Drug treatment is the first-line therapy for BPH,and clinical data show that many patients rely on medication for life to relieve symptoms.There are three kinds of drugs in the domestic and overseas for the treatment of benign prostatic hyperplasia,includingα₁ adrenergic receptor antagonists,5α-reductase inhibitors,plant preparations.While,α₁ adrenergic receptor（α₁-AR）blockers are currently the first-line treatment for benign prostatic hyperplasia.It eliminates smooth muscle contraction caused by dynamic component by blockingα₁-ARs distributed on the smooth muscle of the prostate and bladder neck to relieve bladder outlet obstruction and relax lower urinary tract irritation symptoms.Three differentα₁ adrenergic receptors are expressed in the prostate,namely,α_1A,α_1B andα_1D.It is reported that using onlyα_1A subtype antagonists such as prazosin,terazosin and alfazosin can not effectively improve lower urinary tract symptoms（LUTS）.However,antagonizing the stimulating effect ofα_1D subtype on detrusor dysfunction can relieve the symptoms of perfusion excretion and BPH,which makes up for the lack of high selectivity ofα_1A subtype antagonist drugs.In addition,α₁-AR antagonists not only have effects on the urinary system and relieve BPH symptoms,but also act onα_1B receptor subtypes in cardiovascular system and central nervous system（CNS and CVS）,causing cardiovascular side effects such as orthostatic hypotension、fatigue、sleepiness and sexual dysfunction.Therefore,α_1D/1A subtype selective antagonists with high urinary tract selectivity have been become a research hotspot the development of highly effective and low side effect drugs.Research objective:Naftopidil（NAF）is known as aα_1D/1A selective receptor antagonist,mainly blockingα_1D subtype.It is widely used in clinical treatment of lower urinary tract symptoms caused by BPH.In previous work,our research group designed and synthesized more than 100 arylpiperazine derivatives on the basic of the marketed drug naftopidil（NAF）,and selected two types of new amide antagonist compounds HJZ-3 and HJZ-12 with higher blocking activity and selectivity ofα_1A、α_1Dsubtypes by vitro tissue activity experiment:p A₂ forα_1A were 8.10±0.14 and8.13±0.01,respectively,p A₂ forα_1D were 8.28±0.09 and 8.56±0.06,respectively,while p A₂ forα_1B were 6.29±0.03 and 6.85±0.01,respectively;the selectivity of these two compounds to theα_1D andα_1A subtypes were increased to97.7 and 64.6 times,47.9 and 19.1 times,respectively.However,vivo studies had not been conducted.In addition,in preliminary experiments,we found that HJZ-12had significant cytotoxic activity on human prostate epithelial proliferative cells（BPH-1）and induces apoptosis,but had no effect on the cell cycle;while HJZ-3 had no effect on cells,which might be related to their own chemical structure.Therefore,in this paper,the anti-BPH effect of HJZ-3 and HJZ-12 were fistly studied by estrogen/androgen-induced rat benign prostatic hyperplasia model in vivo;secondly,we tried to explore the effect of pro-apoptotic by HJZ-12 in vitro BPH-1cell model,preliminary analysis of the relationship between the effect of HJZ-12 on BPH-1 cells andα₁ adrenergic receptor.Through the study on the inhibition of BPH by two compounds in vivo and in vitro experiment,we hope to preliminarily explicit mechanism of action and find out new targets.It is necessary to provide scientific basis for further targeted rational design、synthetic drug molecules and in-depth understanding of BPH diseases.Contents and methods:1.Correlation study between HJZ-3,HJZ-12,BPH pathological model of prostate wet weight index,volume index and histopathological changes（1）Establish BPH animal model induced by estrogen/androgen.The sham-operation group and model group were injected normal saline as a control.To study the effect ofα₁ receptor antagonists HJZ-3 and HJZ-12 on BPH model,drug groups were intragastrically administered with positive control NAF（10 mg/kg）、HJZ-3 and HJZ-12（1 mg/kg、3 mg/kg、10 mg/kg）.After 4 weeks,the animals were sacrificed,and the prostate tissue wet weight,volume of each group of rats were weighed.（2）Half of the ventral lobes of the prostate were taken from each group of rats to make pathological sections,HE staining was used to observe tissue morphological changes,and immunohistochemical quantification was performed to investigate the thickness ofα-smooth actin（α-SMA）positive cell layer.Changes were analyzed by Image Pro Plus 6.0 analysis system for tissue morphology.The remaining leaves were stored at-80℃,and the expressions of tissue proliferating cell nuclear antigen（PCNA）protein、apoptotic protein andα_1A、α_1D receptors proteins were detected by Western blot.2.To study the effect of pro-apoptotic by HJZ-12 in vitro BPH-1 cell model and preliminary explore the correlation the relationship between the effect of HJZ-12 on BPH-1 cells andα₁ adrenergic receptor.（1）Cell viability assay:The effect of HJZ-12 on the viability of BPH-1 cells was detected using CCK8 assay;（2）Cell cycle detection:The effect of HJZ-12 on BPH-1 cell cycle was detected by flow cytometry;（3）Apoptosis detection:The effects of HJZ-12 on BPH-1 cell apoptosis were detected by flow cytometry and Western blot;（4）RNA-seq data analysis of possible apoptosis targets of HJZ-12 on BPH-1cells,and verification of changes in m RNA and protein levels by QPCR and Western blot;Western blot was used to detect the expression of the target protein after silence,and the changes of its apoptosis were detected by flow cytometry.At the same time,when silence protein was given in the presence of HJZ-12,we futher examined the change of protein expression and cells apoptosis.（5）Detect whether the effect of HJZ-12 on cell viability and apoptosis were related to theα₁ receptor after intervention with irreversible inhibitor phenbenzamine andα₁ agonist norepinephrine.Research results:1.In vivo study（1）Change of prostate wet weight,volume and relative index in each groupThe prostate wet weight,wet weight index,volume,and volume index of model rats were significantly increased compared with the sham operation group（P<0.001）.In comparation with the model group,the wet weight index of rat prostate was reduced in different dose groups of HJZ-3 and HJZ-12（P<0.05）;and different from NAF,middle,high concentrations of HJZ-3 and HJZ-12 had significant statistical difference in volume index（P<0.05）.（2）Histopathological and quantitative analysis of prostate tissues in each groupIn the sham operation group,the glandular cavity of the prostate was irregular,and the glandular epithelial cells were arranged in a single layer columnar,the glands were surrounded by connective tissue;in the model control group,the glandular epithelium of prostate acini showed uneven hyperplasia.The contents of the lumen were increased,and the epithelial cells were markedly thickened（P<0.001）;the volume of prostate gland lumen was decreased in NAF group;HJZ-3 and HJZ-12 high dose groups were the most obvious changed,prostate gland bubbles were backed to normal,the arrangements were cleared,and the sizes were uniformed.Compared with the model group,each dose group of HJZ-3 had an effect on the glandular epithelium thickness of the prostate.Medium and high doses also had an effect on the volume of the glandular stroma,epithelium and glandular cavity.The inhibitory effect of high dose of HJZ-3 on the relative volume of glandular cavity and thickness of glandular epithelium were better than the corresponding dose of the positive drug NAF（10 mg/kg）.While each dose of HJZ-12 had an effect on the glandular epithelium thickness and glandular cavity volume of the prostate,and the inhibitory effect of high dose on lumen relative volume,epithelial volume and epithelial thickness of prostate gland were also better than the corresponding dose of positive drug NAF（10 mg/kg）by morphometric analysis.（3）Quantitative immunohistochemical analysis ofα-SMA protein in prostate tissues of each groupThe thickness of theα-SMA positive cells layer were measured,immunohistochemical showed that the positive layer around the acinar of the model group was increased significantly in comparison to the sham group（P<0.001）.NAF and each dose of HJZ-3 reduced the thickness of theα-SMA positive cells layer（P<0.01）;while the middle and high doses also exhibited obvious inhibition（P<0.001）.（4）Expression of PCNA and apoptotic proteins in prostate tissues of each groupPCNA protein expressions in prostate tissue were detected.Western blot showed that the PCNA expression level of the model group was increased compared with the sham group.NAF and each dose of HJZ-3 reduced the expression of PCNA（P<0.01）,which did not affect the apoptotic protein（P>0.05）.Differ in NAF and HJZ-3,middle and high doses of HJZ-12 not only reduced the expression of PCNA,but also the middle dose of HJZ-12 could significantly increase the expression of the apoptotic protein Cleaved Caspase-3（P<0.001）and TUNEL-positive cell rate（P<0.01）;while low and high doses of HJZ-12 had no effect on tissue apoptosis（P>0.05）.（5）Expression ofα₁ receptor protein in prostate tissues of each groupThe expression of theα_1A andα_1D receptors proeins in prostate tissue were also detected.The results showed that,compared with the sham operation group,the proteins expressions ofα_1A andα_1D receptors were increased in the model group;theα_1A andα_1D receptors proeins of NAF group were lessened（P<0.01）.HJZ-3 and HJZ-12 decreased the expression ofα_1D receptor proteins（P<0.01）;however,the low,middle doses of HJZ-3 and HJZ-12 had no effect on theα_1D receptor proteins（P>0.05）,high doses of HJZ-3 and HJZ-12 showed a significant reduction inα_1Areceptor expression（P<0.01）.These further confirmed that HJZ-3 and HJZ-12 were acted asα_1D/1A blockers in vivo.2.In vitro study（1）Effect of HJZ-12 on human prostate epithelial proliferative cells（BPH-1）cell viabilityDifferent concentrations（0-100μM）of HJZ-12 affected in BPH-1 cells for 24h and 48 h.HJZ-12 could inhibit cell different degrees of proliferation with concentration-and time-dependent manner.The IC₅₀ values for 24 h and 48 h were23.96±1.05μM and 14.76±2.35μM,respectively.（2）HJZ-12 had no effect on cell cycle of BPH-1 cellsHJZ-12 at different treatment concentrations（0,5,10,15μM）were applied to BPH-1 cells for 24 h.The cells were analyzed with flow cytometry.The results indicated that HJZ-12 had no significant effect on cell cycle.（3）HJZ-12 induced apoptosis in BPH-1 cellsHJZ-12 at different treatment concentrations（0,5,10,15μM）were applied to BPH-1 cells for 24 h.The cells were analyzed with flow cytometry.The results showed that Annexin-V/PI positive rate of HJZ-12（10,15μM）were increased.At the same time,Western blot also showed that the expression of Cleaved Caspase-3proteins were significantly increased.（4）HJZ-12 might induce BPH-1 cells apoptosis by reducing Bmi-1（B lymphoma Mo-m LV insertion region 1 homolog）protein expressionBy analyzing the RNA-seq data,factors related to the apoptotic pathway were selected.The consistency of m RNA levels and protein expressions were verified by QPCR and Western blot.Finally,Bmi-1 factor was selected.After different concentrations（0,5,10,15μM）of HJZ-12 for 24 h in BPH-1 cells,Bmi-1 m RNA levels reduced.At the same time,Western blot showed that HJZ-12 also exhibited a concentration-dependent decrease in Bmi-1 expression level.Furthermore,Bmi-1si RNA was transfected into BPH-1 cells,Western blot was used to detect Bmi-1protein expression.The results showed that after knocking out Bmi-1,the apoptotic rate increased by flow cytometry.Treatment of HJZ-12（15μM）on the basis of Bmi-1-si RNA,the apoptotic rate was significantly increased compared with that of HJZ-12 alone.（5）The relationship between the effect of HJZ-12 on cell viability,apotosis andα₁ adrenergic receptor.First,we assessed cell viability.Different concentrations（0-50μM）)ofα₁receptor agonist norepinephrine（NE）were treated on BPH-1 cells.After 24 h,CCK8 assay showed that NE（0-30μM）could stimulate cell growth,and the pro-growth ability decreased after 30μM.At the same time,30μM NE and HJZ-12co-treated on BPH-1 cells for 24 h,which did not affect HJZ-12 on cell viability.Second is the evaluation of apoptosis.Treatment with theα₁ receptor irreversible inhibitor phenoxybenzamine（1μM,pre-incubate 4 h）and various concentrations of HJZ-12 on BPH-1 cells.After 24 h,Western blot showed that the expression of Cleaved Caspase-3 proteins of HJZ-12（15μM）were significantly upregulated,however,phenylbenzymin reversed the expression of Cleaved Caspase-3 proteins by 10μM of HJZ-12,which indicated that the effects of HJZ-12on BPH-1 cell apoptosis were played by both low-dose dependentα₁-AR and high-dose independentα₁-AR mechanisms.Conclusion:1.In vivo experiment with rat BPH model induced by estrogen/androgen,HJZ-3 and NAF might relieve BPH symptoms by relaxing smooth muscle tension and inhibiting prostate tissue proliferation without inducing apoptosis;HJZ-12might alleviate BPH symptoms in vivo by inhibiting both proliferation and pro-apoptotic effects,which were worthy of further study.2.In vitro,HJZ-12 also showed a better pro-apoptotic effect in the BPH-1 cell experiment.Preliminary mechanism researched HJZ-12 might play a pro-apoptotic role by reducing the level of Bmi-1 protein expression;meanwhile,the effect of HJZ-12 on the viability of BPH-1 cells was independent ofα₁-AR mechanism,the role of apoptosis were low-dose dependentα₁-AR and high-dose independentα₁-AR mechanisms.