The Scientific Basis for the New Application of Cistanche
Mar 09, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Sun Weidong1, Chen fei2, Sun yun2
(1. Yangzhou Hospital of Traditional Chinese Medicine, Yangzhou 225009, Jiangsu; 2. School of Medicine, Yangzhou University, Yangzhou 225001, Jiangsu)
ABSTRACT: The beg nin 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 ultra microstructure prostate gland 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 prostate apoptosis in the model group, in the high-dose acteoside from Cistanche tubulosa treatment, rat prostate apoptosis was significantly higher than that of the model.
KEYWORDS: Cistanche, Cistanche tubulosa, acteoside; rat; begin prostatic hyperplasia; apoptosis; ultrastructural
In the process of benign prostate hyperplasia (BPH), cell apoptosis is one of the mechanisms that are currently attracting attention. Gene coding determines it and enables cells to control programmed cell death by responding to specific signals.
After the prostate develops to adult size, the proliferation rate of prostate cells is maintained at 1% to 2% through the balance of the average rate of apoptosis. Once the balance is broken, it is considered to be the cause of BPH. Under normal conditions, the levels of estrogen and androgen and normal circulation in the prostate are a kind of self-balance state that exists in the process of proliferation and apoptosis. When the balance between proliferation and apoptosis is out of balance, it will lead to further growth and expansion of the gland, which mainly causes the expansion of the epithelial cell compartment [1].
In this experiment, we established a rat prostate hyperplasia model to observe and analyze the effect of acteoside (coaction-like glycoside), one of the chemical components of Cistanche on the apoptosis and ultrastructural changes of rat prostate cells, in order to evaluate the effect of acteoside The effect of inhibiting prostate hyperplasia provides a scientific basis for the development and utilization of new uses of Chinese medicine Cistanche.
Cistanche extract
1 Material and Methods
1. 1 Experimental Animal
Fifty male SD rats aged 6-7 weeks, clean grade, weighing 80-100 g, are provided by the Center for Comparative Medicine of Yangzhou University.
1. 2 Reagents and Medicines
Testosterone propionate injection is from Shanghai General Pharmaceutical Co., Ltd., the specification is 25 mg·mL-1, and the batch number is 060708. PI dye solution is from Yangzhou University Testing Center. The content of Aktiside is 945 g·kg-1, prepared by the Department of Chinese Medicine Analysis, China Pharmaceutical University.
1. 3 Experimental Equipment
Philips Tecnai 12 transmission electron microscope is from Philips in the Netherlands; FACSAria flow cytometer is from BD in the United States.
1. 4 Groupings and Modeling
After castration according to the method of literature [3], rats were injected with 5 mg·(kg·d)-1 of testosterone propionate on the back of the rats for 4 weeks to establish a prostate hyperplasia model. The model rats were randomly divided into model groups: model plus acteoside high-dose group, model plus acteoside medium-dose group, model plus acteoside low-dose group, ten rats in each group; another ten normal rats were as a control group.
1. 5 Administration Method
One week after modeling, acteoside high, medium, and low dose groups were given 60, 30, 15 mg·(kg·d)-1 by gavage, respectively. The model group and the control group were put an equal volume of normal saline once a day for four weeks, weighed once a week, and adjusted the dosage according to the body weight.
1. 6 Sample collection and Processing
1) Material and sample preparation: 24 hours after the last administration, weigh the fasting rats and sacrifice the rats by cervical dislocation. Immediately open the lower abdomen, peel off the tissues around the prostate, free the prostate, and take part of the prostate for use in an ice bath. Tissue homogenizer homogenized, prepared prostate single-cell suspension, and stained with propidium iodide one-step method [4]; another part of prostate tissue was fixed in 25 g·L-1 glutaraldehyde to prepare ultra-thin sections.
2) Prepare a single cell suspension: Under aseptic conditions, remove the rat prostate completely and move it into a petri dish, wash the blood and blood clots on the surface of the prostate tissue with PBS, and remove the surrounding fat, excess fibers, and cell membranes. From the same rat prostate, fresh prostate tissue with the size of about 0.5 cm3 was taken and cut into pieces to prepare a prostate cell suspension [5]. Place the prostate tissue block in a petri dish, and add a small amount of PBS; use ophthalmological scissors to cut the tissue in an ice bath, grind the homogenizer to a suspension, add 10 mL PBS, pipette the tissue homogenate, and use 0.074 mm pore size nylon mesh was filtered into the test tube, centrifuged at 1 500 r min-1 for 3 min, and the precipitate was washed three times with PBS, each time at 500 r min-1 short-term low-speed centrifugation to remove cell debris, to 0.037 mm pore size nylon mesh filter to remove cell clumps. Count the cells and adjust the cell concentration to 2×109 cells·L-1. Stain with 1 mL PI staining solution, protect from light at 4 ℃ for 10 min.
3) Ultrathin section preparation: For ultrathin tissue section preparation, see literature [6].
2 Results and analysis
Fig. 1 Apoptosis of the BPH rat model and induced action of acteoside
a. Normal group; b. Model group; c. Acteoside high-dose group; d. Acteoside low-dose group
2. 1 The effect of acteoside on apoptosis of rat prostate
Figure 1 shows the peak of apoptosis detected by flow cytometry, which shows that acteoside has a pro-apoptotic effect. The statistical analysis of the cell apoptosis results showed that the rat cell apoptosis rate was significantly increased (24.7±1.85)% and (16.1±1.04)% after treatment with high, medium, and low doses of the acteoside. , (14.5±0.68)%, and the normal group (1.1±0.06)%, model group (9.5±0.5)%, positive group (9.83±0.76) The difference in% comparison is significant (P <0.01), indicating that Aktidine can induce cell apoptosis, the effect is stronger than that of the positive control drug Qianliekang (Figure 2).
Fig. 2 The radio of apoptosis in the BPH rat model and effection of acteoside
2.2 The effect of acteoside on the ultrastructure of prostatic hyperplasia cells.
Electron microscopy observations show that the normal rat prostate has complete nuclei, obviously nucleoli, and uniform nuclear chromatin distribution; the rough endoplasmic reticulum has the same size, is neatly arranged, and has no vacuolation. There are no finger-like microvilli on the surface of the epithelium, no swelling of mitochondria, and few secretions in the cavity. The nuclei of prostate cells in the model group were deformed and shrunk, and the cytoplasm was abundant. The rough endoplasmic reticulum was expanded in a cyst-like shape, and the cyst was filled with high-density protein particles. The mitochondria were slightly swollen (there were also vacuoles), There are medium-density secretions in the cavity, and dense round secretion granules were found in some cells. In rats treated with acteoside, the nucleus was intact compared to the model group. The nucleus was concentrated, the nuclear density was increased, heterochromatin accumulated, the cytoplasm had vacuoles, and the rough endoplasmic reticulum vacuoles were reduced. The low-dose acteoside group had slightly wrinkled nuclei Shrinkage, irregular, richer cytoplasm, and reduced rough endoplasmic reticulum (Figure 3).
The benefit of cistanche: anti-Apoptosis
3 Discussion
Apoptosis is a process of programmed cell death in multicellular organisms that regulates the development of the body and maintains the stability of the internal environment, and genetic codes determine it.
Apoptotic cells have their unique morphological and biological characteristics: nuclear chromatin shrinkage, DNA extensive degradation and fragmentation, full-scale shrinkage of cells, shrinkage of cell membranes and outer protrusions covering organelles (such as mitochondria and ribosomes), and the formation of characteristic nuclear fragments Apoptotic bodies, apoptotic cells not only retain a complete cell membrane structure and function but also have a complete structure of organelles, which is different from cell necrosis.
The molecular mechanism of cell apoptosis has not been fully elucidated so far. Studies [7] have shown that there are many genes involved in the gene regulation of cell apoptosis.
Apoptosis is the process of BPH is currently attracting more attention. It is determined by gene coding and enables cells to control cell death by responding to specific signals [1]. After the prostate develops to adult size, the proliferation rate of prostate cells is maintained at 1% to 2% through the balance of apoptosis rate. The destruction of this balance has always been considered as the cause of BPH [8]. Under normal conditions, the level of estrogen and androgen and regular circulation in the prostate are a kind of self-balance state that exists in the process of proliferation and apoptosis. When the proliferation and apoptosis are out of balance, it will cause the glands to grow and expand further, mainly due to the expansion of the epithelial cell compartment [9]. The interruption of this balance may be due to changes in androgen levels or functions; it may also be due to any response produced by AR stimulation of DHT or changes in DHT-mediated growth factor levels [2]. In this study, FCM detected cell apoptosis and found that the apoptosis rate of prostate cells in rats given high doses of acteoside increased significantly. Electron microscopy results showed that the nuclei of prostate cells in the model group were deformed, the rough endoplasmic reticulum was highly expanded in a cyst-like shape, and the cyst was filled with high-density protein particles, and vacuolation was found. The mitochondria were mildly swollen, and related studies[ 10-11 ] The report is consistent.
After acteoside intervention, the degree of prostate hyperplasia in rats was lower. The high-dose group also showed early signs of apoptosis: the nucleus of prostate cells remained intact, and endoplasmic reticulum vacuoles were significantly lower. Condensation of nuclei, increased nuclear density, accumulation of heterochromatin, similar to the characterization of prostate tissue cells in rats after castration [12-13]. It shows that acteoside from the cistanche can induce cell apoptosis in rats with prostate hyperplasia, regulate and restore the balance of prostate cell apoptosis and proliferation, and play a role in inhibiting prostate cell proliferation. The results of this study and previous studies [14] indicate that the inhibitory effect of acteoside, a chemical component of Cistanche tubulosa, on prostate hyperplasia may be related to its promotion of cell apoptosis, reduction of AR and TGF-UR1 overexpression. The detailed mechanism of action still needs to be further studied.
Cistanche benefit: improve immunity
References:
[1] Giacomo N, Antonio G, Ra fa el B B, et al. Inflammation, apoptosis, and BPH, what is the Evidence [J]. European Urology Supplements, 2006(5): 401-408.
[2] Car son I C, Rittmaster R. The role of dihydrotestosterone in benign prostatic hyperplasia [J]. Urology, 2003, 61: 2-7.
[3] Xu Shuyun, Bian Julian, Chen Yuxiu. Pharmacological experiment methodology[M]. 3rd edition. Beijing: People's Medical Publishing House, 2002: 1552-1553.
[4] Zhai Qiwei, Ji Hongbin, Yan Mingda, et al. Using a tet-off inducible expression system to study the function of Akt[ J]. Chinese Science Bulletin, 2000, 45( 16): 1738-1741.
[5] Xia Anzhou, Xing Shuhua, Zhu Xuewen, et al. Comparative study on the preparation methods for renal tissue single-cell suspension [J]. Journal of Xuzhou Medical College, 2004, 24( 1):50-53.
[6] Sun Yun, Wang Dejun, Liu Xiaomei, et al. The changes of lung ultrastructure in experimental aging mice and the effect of Cistanche deserticola polysaccharide[ J]. Chinese Pharmacological Bulletin,2002, 18( 1): 84-87.
[7] Liu Qingming, Lu Mingzhi. Research progress in the pathogenesis of benign prostatic hyperplasia cell proliferation and apoptosis[ J]. Jiangxi Medical Laboratory Science, 2005, 23( 2): 141-143.
[8 ] Sharia t S F, Ashfaq R, Roehrborn C G, et al. Expression of survivin and apoptotic biomarkers in benign prostatic hyper plasma [J]. J Urol, 2005, 174: 2046-2050.
[9 ] Unter G, Madersbacher S, Berg er P. Benign prostatic hyperplasia: age-related tissue-remodeling [J ]. Exp Gerontol, 2005, 40: 121-128.
[10] Zheng Heng, Shao Chunli, Qian Jiaqing. The inhibitory effect of hydrochloride on prostate hyperplasia in rats[ J]. Journal of Tongji Medical University, 1999, 28( 3):227-229.
[11] Zhong Wei, Qiu Shudong, Jiang Sailing. The morphological changes of benign prostatic hyperplasia and the difference of ARmRN A expression [J]. Journal of Xi’an Medical University, 1999,20( 2): 145-149.
[12] Niu Yuanjie, Dong Kequan, Bai Jingwen, et al. Dynamic pathological changes of canine prostate after castration[ J]. Chinese Journal of Urology, 1998, 19( 7): 429-433.
[13] Ye Zhangqun, Lan Ruzhu, Zhou Yusheng, et al. Dynamic changes in the microstructure of the ventral prostate of the rat after castration[ J]. Chinese Journal of Urology, 2001,22( 8): 468-472.
[14] Sun Weidong, Chen fei, Sun yun. The inhibitory effect of Aktiside on experimental benign prostatic hyperplasia model[ J]. Journal of Yangzhou University: Agriculture and Life Sciences
Edition, 2008, 29(3): 55-58.
