The Pharmacological Research Of Acteoside On The Action Inhibited Begin Prostatic Hyperlasia in Rats

The pharmacological research of acteoside on the action inhibited begin prostatic hyperplasia in rats

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ABSTRACT: The begin prostatic hyperplasia (BPH) rat model was established, and three dose groups of acteoside were orally administrated for 4 weeks, to study the effect of acteoside distilled from Cistanche tubulosa on apoptosis and ultramicro structure prostate land in rats with BPH. The ultrastructural change of the prostate gland was observed by transmission electron microscopy. Results showed that Apoptosis of normal rat prostate was not obvious, there was a low rate of pro-state apoptosis in the model group, in the high-dose acteoside treatment, rat pro-state apoptosis was significantly higher than that of the model group. Acteoside could promote rat prostate proptosis, and play ed a role in inhibiting benign prostatic hyperplasia

KEYWORDS: acteoside, Cistanche, begin prostatic hyperplasia

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Apoptosis during prostatic hyperplasia (BPH) is one of the currently more concerning mechanisms, which is genetically encoded and enables cells to control cell death by responding to specific signals. After the prostate develops to adult size, the proliferation rate of prostate cells is maintained at 1% - 2% by the balanced action of the average rate of apoptosis, and once this balance is broken it is considered to be the predisposing cause of BPH. Under normal conditions, the levels of females and androgens in the prostate gland and its normal circulation are a state of homeostasis existing between proliferation and apoptosis. When proliferation is out of balance with apoptosis, it leads to further growth and enlargement of the gland, mainly allowing expansion of the epithelial intercellular compartment to occur. In this test, we observed and analyzed the effect of one of the chemical components of Cistanche, acteoside (like phyllode), on apoptosis and ultrastructural changes in the rat prostate by establishing a rat model of prostate hyperplasia, to evaluate the utility of acteoside in inhibiting prostate hyperplasia, and to provide a scientific basis for developing a new use of Cistanche, a Chinese medicine.

1 material and methods

1. 1 test animals

Fifty male SD rats aged 6-7 weeks, clean grade, weighing 80-100 g, were provided by the comparative medicine center of Yangzhou University.

1. 2 reagents and drugs

Testosterone propionate injection was produced by Shanghai General Pharmaceutical Co., Ltd., specification 25 mg · ML - 1, lot 060708. PI dye solution was provided by the testing center of Yangzhou University. acteoside content 945 g · kg - 1, prepared in the Department of traditional Chinese medicine analysis, China Pharmaceutical University.

1. 3 experimental apparatus

Philips TECNAI 12 transmission electron microscope Dutch Philips; Facsaria flow cytometer manufactured for Becton Dickinson.

1. 4 groupings and modeling

The prostate hyperplasia model was established by subcutaneous injection of testosterone propionate in the back of rats for 4 weeks according to the literature method. The model rats were randomly divided into four groups: model group, model acteoside high-dose group, model acteoside medium-dose group, model acteoside low-dose group, with 10 rats in each group; Another 10 normal rats were taken as a control group.

1. 5 methods of administration

One week after modeling, the rats in the high -, medium -, and low-dose groups received 60, 30, and 15 mg · (kg · d) - 1 by gavage, respectively. The model and control groups were intragastrically administrated with equal volume normal saline, once a day for 4 weeks, and the bodyweight was weighed once a week to adjust the dosage according to the body weight.

1. 6 sample collection and processing

1) Samples and preparation: 24 hours after the last feeding, the bodyweight of the fasting rats was weighed, and the rats were sacrificed by cervical dislocation, and the lower abdomen was immediately opened and the surrounding prostatic tissue was removed, and the free prostate was removed, and some of the prostates were homogenized in a tissue homogenizer in an ice bath to prepare single-cell suspensions of the prostates, which were stained with propidium iodide in a one-step procedure; Another portion of prostate tissue was fixed in 25 g · L - 1 glutaraldehyde, and ultrathin sections were prepared.

2) Preparation of single-cell suspensions: the intact rat prostate was removed into a container under sterile conditions, the blood and blood clot on the surface of the prostate tissue was rinsed with PBS to remove surrounding collateral fat and excess fibrous and cytoplasmic membranes, and a fresh prostate tissue approximately 0.5 cm3 in size was harvested from the same rat prostate, minced, and a prostate cell suspension was prepared.

A block of prostate tissue was placed in a Petri dish and a small amount of PBS was added; The tissue was sheared with ophthalmic scissors in an ice bath, homogenizer ground to suspension, 10 ml PBS was added, the tissue homogenate was aspirated by pipette, filtered into test tubes with 0.074 mm pore size nylon mesh, centrifuged at 1500 R · min - 1 for 3 min, and the pellet was washed three more times with PBS, each time with 500 R · min - 1 short-time low-speed centrifugation to remove cell debris, and 0.037 mm pore size nylon mesh filtered to remove cell clumps. Cells were counted and adjusted to a cell concentration of 2 × 109 · L - 1. Stain with 1 ml PI dye solution for 10 min at 4 ° C in the dark.

3) Ultrathin section preparation: ultrathin tissue section preparation.

2 results and analysis

It is known from the results that acteoside has a proapoptotic effect. Statistical analysis of the results of cell apoptosis showed that the apoptotic rate of rats treated with high, medium, and low doses of acteoside respectively increased significantly, and the difference compared with the normal group, the model group, and the positive group was extremely obvious. It indicated that acteoside could induce cell apoptosis, and the effect was significantly stronger than that of the positive control drug alprostadil.

The effect of acteoside on the ultrastructure of prostate hyperplasia cells was shown by electron microscopy: in normal rat prostate, visible nuclei are intact, nucleoli are obvious, and the nuclear chromatin distribution is even; The rough endoplasmic reticulum was consistent in size, arranged in orderly, no vacuolization phenomenon, no finger-like microvilli protrusions on the epithelial surface, no mitochondrial swelling phenomenon was observed, and there were few luminal secretions. In the model group, the nuclei of prostate cells were obviously deformed and shrunken, and the cytoplasm was abundant; The rough endoplasmic reticulum was highly dilated and markedly vacuolated, and the vesicles were filled with high-density protein granules; Mitochondria were mildly swollen with moderate dense secretions in the lumen and secretory granules were seen in some cells. In rats treated with acteoside, the nuclei were intact compared with those in the model group, with slightly condensed nuclei, increased nuclear compactness, heterochromatin accumulation, some vacuoles in the cytoplasm, and fewer vacuoles in the rough endoplasmic reticulum; In the low-dose acteoside group, the nucleus was mildly shrunken, irregular, and the cytoplasm was more abundant, and the rough endoplasmic reticulum was reduced.

3 Discussion

Quantitative detection of apoptotic cells by FCM is simple, rapid, and objective, and can be performed with multiparametric assays, providing a deeper understanding of changes in apoptosis and its possible regulatory mechanisms.

Apoptosis is a programmed cell death process in multicellular organisms for the regulation of organismal development, and the maintenance of homeostasis determined by gene codes. Apoptotic cells have their unique morphological and biological characteristics: pyknosis of chromatin in the nucleus, extensive degradation of DNA breaks, comprehensive shrinkage of the cell, shrinkage of the cell membrane with outer processes enveloping organelles, and fragments of the nucleus forming characteristic apoptotic bodies, so that apoptotic cells not only retain intact cell membrane structure and function but also organelle structure integrity, which is different from cell necrosis. The molecular mechanism of apoptosis has not been fully elucidated to date, and studies have shown that a variety of genes are involved in the regulation of genes involved in apoptosis.

Apoptosis is currently of greater interest in the process of BPH, which is genetically encoded and enables the cell to control cell death by responding to specific signals. After the prostate develops to adult size, the proliferation rate of prostate cells is maintained at 1% - 2% by the balanced action of the rate of apoptosis, and disruption of this balance has long been considered to be the cause of BPH. Under normal conditions, estrogen to androgen levels within the prostate and normal cycling is a state of homeostasis that exists between proliferative and apoptotic processes. When proliferation is out of balance with apoptosis, it leads to further growth and enlargement of the gland, mainly by expansion between epithelial cell septa. Disruption of this balance may stem from alterations in androgen levels or function; Either response may also arise from stimulation of DHT by the AR or from alterations in growth factor levels mediated by DHT. In this study, FCM was performed to detect cell apoptosis, and it was found that the rate of apoptosis in the prostate of rats given a high dose of acteoside was significantly increased compared with the model group. Electron microscopy showed that the nucleus of prostate cells in the model group was deformed, the rough endoplasmic reticulum was highly dilated in vesicles, the vesicles were filled with high-density protein particles, and vacuolization was obvious; Mitochondria were mildly swollen, consistent with reports from related studies

The degree of prostatic hyperplasia in rats was significantly reduced after acteoside intervention. The high-dose group also developed early signs of apoptosis: the nuclei of prostate cells remained largely intact, and endoplasmic reticulum vacuoles were significantly reduced. The nuclei were condensed with increased nuclear compactness and heterochromatin accumulation, similar to the characterization of cells from post-castration prostate tissue in rats. It was illustrated that acteoside could induce apoptosis in rats with prostatic hyperplasia, regulate and restore the balance between apoptosis and proliferation of prostate cells, and exert an inhibitory effect on the proliferation of prostate cells. The results of the present study and previous studies suggest that the inhibition of prostate hyperplasia by the Cistanche chemical component, acteoside, may be associated with promoting cell apoptosis and attenuating AR and tgf-ur1 overexpression, and the detailed mechanism remains to be fully investigated.