An Investigation On The Anti-liver Cancer Effect Of Cistanche Phenylethanoid Glycosides

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Abstract

Objective: To study the anti-hepatocarcinoma effect of cistanche phenylethanoid glycosides (CPhGs), and to explore its possible mechanism of action. Methods: A mouse liver cancer H22 tumor-bearing model was established by subcutaneous injection into the right axilla of the forelimb. The experiment set up a blank control group, a liver cancer H22 tumor-bearing model group, and a Ganfule positive treatment group (1351.5 mg/kg) with high CPhGs. The dose group (500 mg/kg), the middle dose group (250 mg/kg), and the low dose group (125 mg/kg), a total of 6 groups, each with 10 animals. Except for the blank control group and the model group, the other groups were given Ganfule or CPhGs by gavage for 10 consecutive days. During the monitoring of the general condition of the mice, the growth of tumor bodies of H22 tumor-bearing mice in each group was observed. At the end of the experiment, the eyeballs were taken to collect blood, and the serum interleukin 2 (IL-2) and tumor necrosis factor were detected by enzyme-linked immunosorbent assay (ELISA). a(TNF-∞ and alpha-fetoprotein (AFP) content; separate the spleen and liver, weigh them, calculate the organ coefficient; completely strip the tumor body, calculate the tumor inhibition rate of each group of mice; observe the pathological tissue of the tumor body by HE staining. Results: The axillary tumors of the tumor-bearing model group appeared early and grew the fastest, followed by the low and medium-dose CPhGs group, and the tumors in the high-dose group and the positive treatment group grew slowly. Compared with the model group, CPhGs showed medium and fast growth. The tumor mass in the high-dose group was significantly reduced (P<0.05), and the tumor suppressor rate in each dose group increased with the increase of CPhGs dose (<0.05); the positive treatment group and the spleen coefficient of each dose group of CPhGs increased significantly And liver coefficients were significantly reduced (P<0.05); the levels of IL-2 in CPhGs medium, high-dose groups, and positive treatment groups were significantly increased (P<0.05), while the content of AFP was significantly reduced (, I< 0.05); TNF-0 [content in each dose group and a positive treatment group of CPhGs was significantly reduced (P<0.05). Pathological results showed that the growth of mouse liver cancer cells in each dose group of CPhGs was inhibited, the number was smaller, the heterogeneity was reduced, and a large number of necrotic cells were accompanied. Conclusion: CPhGs can reduce liver damage in H22 tumor-bearing mice and inhibit tumor growth, which may be related to CPhGs reducing the serum AFP content in tumor-bearing mice and improving the immunity of tumor-bearing mice.

[Keywords] Cistanche; total phenylethanoid glycosides; H22 cells; liver cancer

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Cistanche (tubulosa) is mainly produced in Xinjiang and the Alexa League of Inner Mongolia. It is known as the "desert ginseng" and has extremely high medicinal value. It is a precious traditional Chinese medicinal material. First published in "Shen Nong's Materia Medica", it is listed as the top grade. It is sweet, salty, and warm in nature. It has the effects of nourishing kidney yang, nourishing essence and blood, and moisturizing the intestines. It is often used to treat impotence in men, infertility in women, and waist and knees. Cold pain, blood dryness, constipation, and other symptoms ridicule. Domestic scholars have conducted a lot of research on the chemical components of Cistanche, including phenylethanoid glycosides from cistanche (CPhGs), iridoids, lignans, polysaccharides, alkaloids, etc. The most important components are CPhGso, modern pharmacological research has shown that CPhGs have antibacterial, anti-inflammatory, antiviral, anti-tumor, anti-oxidant, immune regulation, memory enhancement, yang, and other pharmacological activities. At the same time, a large number of research team members conducted on CPhGs in the early stage showed that CPhGs It has anti-liver fibrosis effect". 71. In this experiment, CPhGs isolated from Cistanche medicinal materials were used to study its anti-hepatocarcinoma effect on H22 tumor-bearing mice. Provide a basis for drug development.

1. Materials and methods

1.1 The test substance and experimental animals

Cistanche phenylethanoid glycosides (CPhGs) were extracted, separated, and identified by researcher Zhao Jun of Xinjiang Institute of Materia Medica, the purity was 70% 91, and 0.5% carboxymethyl cellulose was used in the experiment. Sodium (0.5% CMC-Na) is dissolved, prepared into different concentrations, stored at 4 ℃, and used within 1 week.

SPF-class Kunming mice, 60 males, weighing 18-22g, purchased from the Experimental Animal Research Center of Xinjiang Medical University, the certificate number is SCXK (new) 2016—0002, and the license number is SYXK (salary:) 2016—0003.

1.2. The cell line mouse

H22 hepatocarcinoma cell line is provided by Wuhan Prose Life Technology Co., Ltd., the item number is CL-01341.

1.3. Apparatus and main reagents

Low-speed centrifuge (70L-5A, Shanghai Anting); CO, incubator (Heal Force, HF240); electric heating constant temperature water tank (DK-8D, Shanghai ◎Heng Technology Co., Ltd.); Microplate reader (Bio-rad); vernier caliper (Shanghai Future Experimental Equipment Co., Ltd.); electronic balance (Changzhou Wantai Balance Instrument Co., Ltd.).

Gainful is a product of CommScope Pharmaceutical Co., Ltd. (Z20060389); RPMI-1640 medium and 1% double antibody (Isher IJBI); Fetal bovine serum (Gibco, USA); 0.4% Trypan blue and 4% paraformaldehyde (Jing Suo Lebao); mouse tumor necrosis factor IX (TNF-cc), interleukin 2 (IL-2), alpha-fetoprotein (AFP) ELISA kit (Wuhan Huamei).

1.4 Test method

1.4.1 Establishment of H22 tumor-bearing mouse model of liver cancer

Resuscitate H22 cells at 37℃, discard the supernatant by centrifugation, add fresh RPMI-1640 complete medium to resuspend the cells for rapid recovery, culture more than 95% of viable cells in vitro, and inoculate mice when the cell density reaches 1×106/mL Abdominal cavity, 0.2 mL each. Passage approximately 8.10 d. The mouse peritoneal H22 tumor fluid cultured to the right of 8 dE was aseptically extracted, diluted with normal saline to adjust the number of tumor cells to 1X 107/mL, mixed well, and set aside. Except that the blank control group was injected with the same amount of normal saline at the same site, the mice in the other groups were disinfected and inoculated with H22 tumor cell suspension on the right armpit of their forelimbs, each o. 2mL [9.101, 24 hours after modeling, observe the tumor growth every day.

1.4.2 Grouping and administration

The H22 tumor-bearing mice modeled above were randomly divided into 5 groups, namely the model group and the positive treatment group (Ganfule 1351.5 mg/kg), with high CPhGs (500 mg/kg), QB (250 mg/kg) mg/kg), low (125mg/kg) dose group, each group has 10 animals. Gavage was started at 24 hA after modeling. Except for the blank control group and the model, the group was given the same volume of 0.5% CMC-Na solution, the other groups were given a given dose of positive drugs or CPhGs by gavage for 10 consecutive 10 hours. d"2.1 31. Interview daily observation of mental activity status, right axillary swelling size and appearance time, coat color, and appetite of tumor-bearing mice. 24 hours after the last administration, weigh the weight, remove the eyeballs and collect blood, and separate the serum Spare; Cervical dislocation method kills mice, separates the spleen and liver, weighs the liver and spleen coefficient; peels off the tumor completely, wipes clean with filter paper and weighs the mass, calculates the tumor inhibition rate of each group of mice; the tumor tissue is pathological Histological examination.

1.4.3 Testing indicators

①Determination of spleen and liver coefficients: After the mice in each group were sacrificed, the spleens and livers were cut and weighed, and the organ coefficients were calculated. Organ coefficient = organ mass/4, mouse body mass × 100%. ②Determination of the tumor mass and tumor inhibition rate: 24 hours after the last administration, the mice were sacrificed by the cervical dislocation method and weighed. The tumor was completely peeled off, wiped clean with filter paper, and then weighed on an electronic balance. The mass was calculated based on the tumor mass. Tumor inhibition rate of mice in each administration group, tumor inhibition rate=(model group tumor mass-experimental group tumor mass) membranous group tumor mass X 100%. ③After removing the eyeballs and taking blood, separate the serum and store it at 80°C for inspection. Detect IL-2, TNF-0, and AFP content in serum according to the ELISA kit instructions. ④Tumor histopathological examination. After the tumors in each group were fixed with 4% paraformaldehyde for 24 hours, the sections were embedded in paraffin and stained with conventional HE. The pathological changes of the tumor were observed under the light microscope, and the area of tumor cell necrosis was preliminarily judged. CPhGs drug treatment Effect.

1.5 Statistical methods

The SPSS 17.0 software was used for statistical analysis, and the data results of each group were expressed as x±S, and the data between multiple groups were compared by one-way analysis of variance, and the test level a=0.05.

2. results

2.1 The general observation of mice

Before administration, there was no statistically significant difference in the bodyweight of the mice between the groups (P>0.05). In the model group, 2 mice died due to gastric administration during the experiment. After administration, compared with the blank control group, the bodyweight of the model group mice increased with the growth of the tumor, but the difference was not statistically significant

(P>0.05); Compared with the model group, the bodyweight of mice in the positive treatment group and the CPhGs dose group decreased to a certain extent, but the difference was not statistically significant (P>0.05). On the 4th day of the experiment, the underarms of the mice began to swell, the animal's state of mental activity became worse, the animals were slow to respond to external stimuli, their appetite decreased, and their coat color became dim. The tumor mass appeared first in the model group, and the growth rate was the fastest. The above reactions were the most obvious, followed by the CPhGs low and medium-dose group. The high dose group and the positive treatment group had slower tumor growth, a better quality of life, and no obvious adverse effects.

2.2 The effect of Cistanche CPhGs on spleen and liver coefficients of H22 tumor-bearing mice

Compared with the blank control group, the spleen coefficient of the model group was significantly reduced and the liver coefficient was significantly increased (P<0.01); compared with the model group, the spleen coefficient of the positive treatment group and the CPhGs dose groups were significantly increased, and the liver coefficient was significantly decreased (P <0.01), indicating that CPhGs can improve the immune function of tumor-bearing mice to a certain extent and inhibit liver damage.

3. Discussion

Primary liver cancer is a common malignant tumor in clinical practice. It accounts for the 4th place in the world's tumor incidence and the 2nd place in mortality. Its prevention and treatment is still a hot research topic in my country. The advantages of traditional Chinese medicine in controlling the development of patients, improving symptoms and signs, and improving the quality of life are gradually emerging. In order to find low-toxic and high-efficiency anti-liver cancer drugs from Chinese traditional medicine, this topic uses H22 liver cancer cells to prepare H22 tumor-bearing cells. Mouse tumor model, compare the different doses of CPhGs treatment group with the model control group and Ganfule positive treatment group to explore the anti-tumor effect of CPhGs on H22 tumor-bearing mice and its mechanism.

The results showed that the model group was the first to have axillary tumor swelling and its growth was the fastest. The mice's mental state decreased, appetite decreased, and the coat color began to dim. The low and medium-dose group of CPhGs followed by the high-dose group and Ganfule positive. The treatment group was slower, and the bodyweight of mice in each dose group of CPhGs and there was no significant decrease in the Ganfule positive treatment group, suggesting that CPhGs drugs can relatively improve the quality of life of H22 tumor-bearing mice and reduce the toxicity of tumors to the body. The tumor quality of each dose group of CPhGs and Ganfule positive treatment group was significantly lower than that of the model control group, and the tumor inhibition rate of the high-dose group was equivalent to that of the Ganfule positive treatment group, suggesting that CPhGs can affect H22 tumor-bearing mice Tumors in vivo have a certain inhibitory effect. In addition, the results of this study show that the spleen coefficients of the Ganfule positive treatment group and the CPhGs dose groups are higher than the model group, while the liver coefficients are lower than the model control group, suggesting that the drug is effective, This project uses H22 liver cancer cells to prepare H22 tumor-bearing mouse tumor models, compares the different doses of CPhGs treatment group with the model control group and the Ganfule positive treatment group, and explores the anti-tumor effects of CPhGs on H22 tumor-bearing mice. Its mechanism.

The results showed that the model group was the first to have axillary tumor swelling and its growth was the fastest. The mice's mental state decreased, appetite decreased, and the coat color began to dim. The low and medium-dose group of CPhGs followed by the high-dose group and Ganfule positive. The treatment group was slower, and the bodyweight of mice in each dose group of CPhGs and the Ganfule positive treatment group did not significantly decrease, suggesting that CPhGs drugs can relatively improve the quality of life of H22 tumor-bearing mice and reduce the toxicity of tumors to the body. The tumor quality of each dose group of CPhGs and Ganfule positive treatment group was significantly lower than that of the model control group, and the tumor inhibition rate of the high-dose group was equivalent to that of the Ganfule positive treatment group, suggesting that CPhGs has a certain effect on tumors in H22 tumor-bearing mice Inhibition. In addition, the results of this study showed that the spleen coefficients of the Ganfule positive treatment group and the CPhGs dose groups were higher than the model group, while the liver coefficients were lower than the model control group, suggesting that CPhGs can inhibit tumors at the same time. To a certain extent, it improves the immune function of tumor-bearing mice and also has a protective effect on the liver, but the effect on the spleen and liver is not significantly different from that of Ganfule.

IL-2 is a type of cell growth factor in the immune system. It participates in cellular immunity and plays a central regulatory role in the complex immune network of the body. It can improve the body's immune function by promoting the proliferation and differentiation of T cells and can produce anti-Tumor active immune cells. Studies have found that IL-2 can promote the survival, expansion, and activation of lymphokine-activated killer cells. This type of cell is a kind of killer cell with high anti-tumor cells produced by lymphocytes in contact with IL-2, and this killer cell Cells only recognize tumor antigens and have no effect on normal host cells" 5.161. The results of this experiment show that the high and medium dose groups of CPhGs can significantly increase the IL-2 content of mice, indicating that CPhGs can enhance the immunity of H22 tumor-bearing mice Ability to help the body kill tumor cells.

TNF-α is one of the biologically active factors with the strongest direct anti-tumor effect found so far"7-81. TNF induces tumor cell apoptosis, enhances host immune function, and acts on tumor vascular endothelial cells by binding to receptors. Vascular dysfunction leads to tumor hemorrhage and necrosis to exert its anti-tumor effect"91, but TNF-α exhibits duality in tumors, which can cause tumor necrosis and promote tumor growth." TNF-α in normal organisms The low concentration of α has the effects of inhibiting tumor cell proliferation, promoting tumor cell blood vessel formation and thrombosis to cause hemorrhage and necrosis, and enhancing the body's anti-tumor immune function. At the same time, TNF is an endogenous tumor-promoting factor and is produced by tumor cells themselves. TNF-α can stimulate tumor cell growth and inhibit the toxic effect of immune effector cells on tumor cells; therefore, the biological effect of TNF-α is closely related to its level in the body. In this experiment, it was found that compared with the normal group, the model group TNF-α increased significantly, which is consistent with the increase of tumor TNF-α. The TNF-α level of mice in each dose group of CPhGs decreased significantly, indicating that CPhGs can inhibit the abnormal increase of TNF-α in the serum of H22 tumor-bearing mice, Inhibit the occurrence of tumors.

Under normal circumstances, AFP mainly comes from embryonic liver cells. AFP disappears from the blood about two weeks after birth. Therefore, the content of AFP in the serum of normal adults is less than 20 gg/mL. twenty-one. In adults, AFP can be elevated in the serum of approximately 80% of liver cancer patients31. When human liver cells become cancerous, they restore the function of producing the protein AFP, and as the disease worsens, its serum content will also increase sharply. As a specific clinical indicator for diagnosing primary liver cancer, AFP concentration in serum is usually positively correlated with the size of liver cancer1. The results of this experiment show that the high and medium dose groups of CPhGs can significantly reduce the content of AFP in mice, indicating that CPhGs can inhibit the expression of AFP in the serum of H22 tumor-bearing mice and inhibit tumor growth.

HE staining shows that compared with the model group, CPhGs can significantly promote tumor cell apoptosis, inhibit tumor cell growth, reduce tumor cell heterogeneity, and are accompanied by a large number of cell necrosis. Among them, CPhGs in different dose groups increases with the dose With increase, the necrotic area of tumor cells is also increasing, and the high-dose CPhGs group and the Ganfule positive treatment group have a considerable amount of tumor cell necrosis, suggesting that CPhGs has a good anti-tumor effect on H22 liver cancer tumor-bearing mice.

In summary, CPhGs have a good anti-liver cancer effect, which may be related to CPhGs reducing the serum AFP content of tumor-bearing mice and improving the immune ability of tumor-bearing mice. This study provides basic data for the development of new anti-liver cancer drugs using CPhGs in Cistanche.

References

[1] Peng Fang, Xu Rong, Wang Xia, et al. Research progress in the processing and processing of medicinal materials of the genus Mesquite [JI Modern Chinese Medicine, 2015, 17(4): 406-412.

[2] Zhong Yaoxin. Interpretation of "Shen Nong's Materia Medica" 32[J], It is beneficial to open the volume (seeking medical advice),

2015, 35(8): 40-41.

[3] Liu Xiong, Li Chengming, Gao Jiande, et al. Research progress of meat ash paste[J]. China Journal of Traditional Chinese Medicine and Technology, 2013, 20(5): 575-576.

[4] Geng Huijie, Yu Guangen. Development and utilization of plants of the genus Sargassum [J]. Tianjin Science and Technology, 2012, 21 (4): 66-68.

[5] You Shuping, Zhao Jun, Ma Long, et al. The effect and mechanism of ethanol extract of meat ash paste on immune liver fibrosis in rats [J]. Chinese Journal of Pharmacology and Toxicology,

2016, 30(5): 504-510.

[6] Mu Ke Remu • Di Mai Ti, You Shu Ping, Zhao Jun, et al. Experimental study on the effect of Phenylethanoid Glycosides oxime from meat ash on BSA-induced liver fibrosis in rats[J]. Journal of Xinjiang Medical University, 2015, 38(5): 567-570.

[7] You Shuping, Zhao Jun, Mu Keremu•Di Mai Ti, et al. Effects of Roucong Rong's Phenylethanoid on the expression of transforming growth factor|31 in rats with BSA-induced liver fibrosis [JL Cancer Transformation. Distortion•Mutation, 2015, 27(6): 409-414.

[8] You Shuping, Zhao Jun, Ma Long, et al. The effect of total meat ash puree phenethyl alcohol on the expression of TGF-01/Smad signaling pathway in rat hepatic stellate cells[J]. Carcinogenesis. Distortion and Mutation, 2016, 28(5): 372-376.

[9] Zhang Jianwu, Pu Qigong, Chen Doujia, et al. Study on the effect of the aspirin-nicotinamide-zinc complex in mice bearing H22 liver cancer［J］. Sichuan Journal of Physiological Sciences, 2012, 34(4): 148-149.

[10] Shen Julio. The anti-tumor effect of Kuiling Huajijian on mouse H22 hepatocarcinoma cells and its influence on the expression of C-my and nm23-Hl [D]. Dalian: Dalian Medical University, 2013: 8-9.

[11] Hao Mingzhi, Chen Wujin, Lin Hailan, et al. Clinical efficacy observation of hepatic artery embolization chemotherapy combined with Ganfule capsule in the treatment of advanced liver cancer［J］. Zhongnan Pharmaceutical, 2013, 11⑵: 147-150.

[12] Guo Shengqi, Huang Tingzhang, Li Yuanhui, et al. Study on the antitumor effect of water extracts from the stems of jasmine on H22 tumor-bearing mice]. Chinese Journal of Clinical Pharmacology, 2015, 31(10): 855-857.

[13] Zhang Wenjuan, Jiang Weizhe, Lan Xianli, et al. Study on anti-tumor mechanism of Wu Gu insect extract on H22 liver cancer cell tumor-bearing mice]. Guangxi Medicine, 2017, 39(2): 215-219.

[14] Ye Shenglong. New progress in the field of liver cancer in 2013［J］. Chinese Journal of Hepatology, 2014, 22(1): 2-4.

[15] Liu Xiaorui, Wang Jian. Progress in the clinical application of cytokine-induced killer cells against tumors［J］. Guangdong Medicine, 2016, 37(18): 2840-2843.

[16] Song Haojie. Low-dose chemotherapy combined with genetically modified dendritic cell vaccine to treat liver

Experimental research on cancer［D］. Guangzhou: Southern Medical University, 2012.

[17] Zuo Shaoyuan, Qian Jintao, Fang Shuhuan, et al. Anti-tumor activity of safflower bee pollen polysaccharide［J］. Frontier of Medicine, 2014, 4(1): 42-43.

[18] Ni Yang. The effect of Lithospermum polysaccharide on immune organs and cytokines TNF-a and IFN-y in S180 tumor-bearing mice［J］. Heilongjiang Medicine, 2014, 27(3): 512-514.

[19] Gu Cuiping, Zhang Yiping. New advances in the anti-tumor mechanism of TNF-a [J]. Chinese Tumor, 2007, 16⑵: 102-105.

[20] Zhu Bo, Zhang Wei, Chen Yanhua, et al. Application of tumor-specific growth factor and tumor necrosis factor in the diagnosis and prognosis of liver cancer［J］. Modern Oncology Medicine, 2008, 16(5): 765-766.

[21] Lin Yu. Preliminary study on the anti-hepatocarcinoma effect and mechanism of spider odoriferous drugs [D]. Chengdu: Southwest Jiaotong University, 2015.

[22] Wang Shimo, Hu Yonghao, Li Qiang, et al. Correlation analysis between AFP level and the prognosis of patients with primary hepatocellular carcinoma surgery［J］. Chinese Journal of Hepatobiliary Surgery, 2017, 23(2): 134-136.

[23] Song Wei. The clinical significance of combined detection of serum AFP, AFU, GP73, and IL-8 in the diagnosis of primary liver cancer [J] Chinese Journal of Clinicians (Electronic Edition), 2016, 10(9): 1266-1270.

[24] Guan Yu, Yu Xinfa, Zhou Chengyu, et al. The clinical value of alpha-fetoprotein in the diagnosis and prognosis of primary liver cancer［J］. Hainan Medicine, 2016, 27(6): 891 894.

RANKL and their effect on bone diseases[J]. Prog Mod Biomed, 2010, 10(20): 3963-3966.

[14]FRANCESCHI R T, XIAO G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways[J]. J Cell Biochem, 2003, 88(3): 446-454.

[15]FRANCESCHI R T, XIAO G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways[J]. J Cell Biochem, 2003, 88(3): 446-454.

[16]GUICHEUX J, LEMONNIER J, GHAYOR C, et al. Activation of p38 mitogen-activated protein kinase and c-Jun- NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation^]. J Bone Mineral Res, 2003, 18(11): 2060.

[17]GE C, CAWTHORN WP, LI Y, et al. Reciprocal control of osteogenic and adipogenic differentiation by ERK/MAP kinase phosphorylation of Runx2 and PPARgamma transcription factors[J]. J Cell Physiol, 2016, 231(3): 587 -596.

[18]GE C, XIAO G, JIANG D, et al. Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor[J]- J Biol Chem, 2009, 284(47): 32533-32543.

[19]GREENBLATT M B, Shim J H, GLIMCHER L H. Mitogen-activated protein kinase pathways in osteoblasts[J]. Annual Rev Cell Dev Biol, 2013, 29(1): 63.

[20] Li Feng, Liu Wenfeng, Liu Rushi, et al. Malondialdehyde inhibits the osteogenic differentiation of mesenchymal stem cells by activating p38 and JNK pathways[J]. Chinese Journal of Biochemistry and Molecular Biology, 2012, 28(9): 804-810.

[21]ZHANG G M. Ubiquitin ligase Smurfl controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation^]. Cell, 2005, 121(1): 101-113.