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1Traditional Medicine Research  2019, Vol. 4 Issue (2): 99-108    DOI: 10.12032/TMR20190304108
Article     
Total saponins in Rubus parvifolius L. induce lymphoma cells apoptosis through upregulated Bax/Fas and downregulated Bcl-2 in vivo and in vitro
Xiao-Feng Xu1,*(), Ru-Bin Cheng2, Xue-Jin Zhang1, Rui-Lan Gao3
1 Department of Hematology, Zhejiang Provincial Integrated Chinese and Western medicine Hospital, Hangzhou, China.
2 College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, China.
3 The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China.
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Highlights

Total saponins of Rubus parvifolius L. (TSRP) induces Raji cell apoptosis by inhibiting Bcl-2, increasing Bax expression, and reducing Fas expression in vivo and in vitro. Additionally, TSRP has some effects on the chondriosome pathway of apoptosis in vitro.

Traditionality

Rubus parvifolius L. (RP) belongs to the family Rosaceae and is mainly produced in the temperate zone of the Northern hemisphere. As early as the Xihan Dynasty of China, Erya recorded that RP were edible. Bencao Shiyi recorded the medicinal properties of the root bark and fruit of RP, which include clearing heat, cooling blood, stopping bleeding, dispersing knots, relieving pain, diuresis, and detumescence.

Abstract

Purpose: To investigate the effect of total saponins of Rubus parvifolius L. (TSRP) on lymphoma Raji cells and further discuss its mechanism. Methods: The model of nude mice bearing Raji cells was established, the volume, weight and inhibition rate of the transplanted tumor were analyzed and compared after different concentrations of TSRP treatment. Cell apoptosis and expression of Bcl-2, Bax, Fas proteins were detected by TUNEL and immunohistochemiscal method respectively. Effects of TSRP on cell proliferation were tested with MTT assay in vitro. Cell apoptosis and expression of Caspase-9, Caspase-3, Bcl-2, Bax and Fas proteins were tested with DAPI staining and Western blot. Results: TSRP significantly reduced the volume and tumor weight of Raji subcutaneous transplanted tumor and induced the apoptosis of Raji cells in vivo. The tumor inhibition rate of high-dose (100 mg/kg) TSRP is 90.84%. The TUNEL test results show that the fluorescence intensity of the tumor issue treated with TSRP is significantly improved. Compared with the control group, the fluorescence intensity of high-concentration TSRP is 82.43 ± 7.81, which is significantly different (P < 0.001). The results of immunohistochemistry test showed that the Bcl-2 expression of Raji cell treated with TSRP is obviously reduced, and Bax expression is obviously increased. Meanwhile, compared with that of control group, Fas expression is obviously reduced. MTT assay showed that TSRP can significantly inhibit proliferation of Raji cells with dose dependence. The inhibition rate of 400 μg/mL TSRP is 53.46 ± 4.90% (P < 0.001). DAPI staining results showed that TSRP can significantly induce cell apoptosis. According to Western blot results, it is found that TSRP can significantly inhibit activity of Bcl-2 and increase Bax expression, and TSRP can also inhibit Fas expression. Meanwhile, expression of Caspase-9 and Caspase-3 is also increased. Conclusion: TSRP could inhibit the proliferation of lymphoma via induction of apoptosis in a time and dose-dependent manner. Apoptotic signaling induced by TSRP was characterized by up-regulating Bax, Fas and Caspase-8 protein expression, and down-regulating of Bcl-2 protein expression.



Key wordsTotal Saponins of Rubus parvifolius L      Bcl-2      Bax      Fas      Apoptosis      Lymphoma     
Received: 28 December 2018      Published: 05 March 2019
Fund:  The study were supported by grants from Zhejiang Provincial Administration of Traditional Chinese Medicine (No. 2011ZA081, 2013ZB095 and 2015ZA147), Hangzhou Medical Science and Technology Plan (No. 2012A048).
Corresponding Authors: Xu Xiao-Feng     E-mail: hhxuxiaofeng@126.com.
Cite this article:

Xiao-Feng Xu, Ru-Bin Cheng, Xue-Jin Zhang, Rui-Lan Gao. Total saponins in Rubus parvifolius L. induce lymphoma cells apoptosis through upregulated Bax/Fas and downregulated Bcl-2 in vivo and in vitro. 1Traditional Medicine Research, 2019, 4(2): 99-108. doi: 10.12032/TMR20190304108

URL:

https://www.tmrjournals.com/tmr/EN/10.12032/TMR20190304108     OR     https://www.tmrjournals.com/tmr/EN/Y2019/V4/I2/99

Groups Tumor weight (g) Size (mm3) Tumor inhibition rate (%)
Control 1.34 ± 0.50 2807.70 ± 128.43 -
TSRP (20 mg/kg) 0.86 ± 0.01 2022.59 ± 82.39# 27.96
TSRP (100 mg/kg) 0.21 ± 0.03* 257.29 ± 43.65# 90.84#
Table 1 Comparison of the tumor weight, size and tumor inhibition rate of each group after intragastric administration with TSRP
Groups Intragastric administration time (day)
8 11 14 17 20
Control 307.46 ± 13.83 456.57 ± 15.98 1093.29 ± 125.19 2080.01 ± 112.03 2807.70 ± 128.43
TSRP (20mg/kg) 212.51 ± 18.31 430.76 ± 10.28 998.43 ± 90.76 1122.59 ± 132.39# 2022 ± 182.39#
TSRP (100mg/kg) 207.22 ± 4.84 214.34 ± 8.32 235.45 ± 7.98# 243.15 ± 23.65# 257.29 ± 43.65#
Table 2 Tumor size change of the nude mice after intragastric administration (mm3)
Figure 1 Comparing Raji cell apoptosis of the transplanted tumor of each group with TUNEL analysis
TSRP, Total saponins of Rubus parvifolius L.. #, compared with the control group, P < 0.001.
Figure 2 Expression of Bcl-2, Bax and Fas of the transplanted tumor of each group (×200)
TSRP, Total saponins of Rubus parvifolius L..
Groups Absorbance Inhibition rate (%)
Control 0.780 ± 0.052 -
TSRP (100μg/ml) 0.639 ± 0.024 18.15 ± 3.33
TSRP (200μg/ml) 0.531 ± 0.034 32.21 ± 4.89
TSRP (400μg/ml) 0.366 ± 0.031 53.46 ± 4.90#
Table 3 Effects of TSRP at different concentrations with MTT
Figure 3 Morphology comparison after TSRP induces Raji cell apoptosis with DAPI analysis (fluorescence microscope × 200).
TSRP, Total saponins of Rubus parvifolius L..
Figure 4 Effect of TSRP at different concentrations on activity of Raji cell apoptosis controlling proteins Bcl-2, Bax, Fas, Casepase 9 and Casepase 3
Compared with the control group: #, P < 0.001; *, P = 0.027. TSRP, Total saponins of Rubus parvifolius L..
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