Search JIM Advanced Search

Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (6): 483-494.doi: 10.1016/S2095-4964(14)60057-1

• Research Article • Previous Articles     Next Articles

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

Zhang Yuan-huia,b,c,d, Wang Yuana,b,c,d, Hussein Yusufali Alic,d, Ashby Frederickc,d, Zhang Danielc,d, Yin Zi-feia,c,d, V. Aslanidi Georgec,d,e, Srivastava Arunc,d,e,f,g, Ling Chang-quana,b, Ling Chen3,4,6   

  1. Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai 200433, China
    Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    c Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Genetics Institute, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Shands Cancer Center, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32611, USA
  • Received:2014-09-04 Accepted:2014-09-28 Online:2014-11-10 Published:2014-11-15

Objective

Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted.

Methods

We synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors. 

Results

The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin (TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model. 

Conclusion

Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Key words: Medicine, Chinese traditional, Cytotoxic genes, Trichosanthin, Recombinant adeno-associated virus vector, Cancer gene therapy

"

Figure 1

Schematic outline of used plasmids AAV: adeno-associated virus; AFP: alpha-fetoprotein promoter; bGH(A)n: bovine growth hormone polyA; CBAp: chicken β-actin/cytomegalovirus enhancer hybrid promoter; CMVp: cytomegalovirus promoter; EGFP: enhanced green fluorescence protein; Gluc: Gaussia luciferase; HP: hairpin structure; GOI: gene of interest; hGH(A)n: human growth hormone polyA; HP-: hairpin structure without terminal resolution site; hrGFP: humanized recombinant green fluorescence protein; IRES: internal ribosome entry site; TCS: trichosanthin."

Figure 2

Protein translations from the potential cytotoxic traditional Chinese medicine genes A: Gel electrophoresis showing Sma I-digested and undigested plasmids; B: Fluorescence microscopy results at 48 h post-transfection with indicated plasmids in HeLa cells; C: Western blot assay showing protein expression in HeLa cells."

Figure 3

Expression of cytotoxic traditional Chinese medicine genes results in a reduction in cancer cell proliferation A: Various cancer cell types were transfected with indicated plasmids. The percentage of viable cells, compared to untransfected cells, was determined by CCK-8 kit at 48 h post-transfection. Data are expressed as mean ± standard deviation; **P<0.01, vs IRES-hrGFP. B: Various hHCC cell lines were transfected with TCS-containing plasmids. Viable cell numbers were determined by CCK-8 kit over time. The red numbers indicate the transfection efficiency in various cell lines. The experiment was repeated three times using triplicate samples in each experiment. CCK-8: Cell Counting Kit-8; hHCC: human hepatocellular carcinoma; TCS: trichosanthin."

Figure 4

Effects of intracellular expressed TCS on protein translation A: Fluorescence microscopic images at 48 h post-transfection with indicated plasmids in HeLa cells. B: Expression of TCS gene dramatically decreases Gluc expression (upper panel) at 48 h post-transfection, but not the presence of Gluc mRNA (middle panel) and DNA (lower panel). The experiment was repeated three times using triplicate samples in each experiment. TCS: trichosanthin; Gluc: Guassia luciferase."

Figure 5

Expression of TCS gene induces apoptosis A: Expression of TCS genes in HeLa cells causes an increased percentage of apoptotic cells determined by annexin V/PI double staining followed by FACS analysis. Cells treated with 1 mmol/L H2O2 or etoposide for overnight were used as positive controls. B: Western blot analysis reveals that expression of TCS gene results in changes in mitochondrial pathways in HeLa cells. C: Expression of TCS gene in different human hepatocellular carcinoma cell lines causes an increase in level of cleaved PARP. 1: Mock-treated cells; 2: Cells transfected with pAAV-CMVp-IRES-hrGFP; 3: Cells transfected with pAAV-CMVp-TCS-FLAG-IRES-hrGFP; 4: Cells treated with 1 mmol/L H2O2 for 18 h. The experiment was repeated three times using triplicate samples in each experiment. TCS: trichosanthin; PARP: poly-ADP-ribose polymerase."

Figure 6

Expression of TCS gene reduces growth of hHCC xenograft in NSG mice Huh7-Fluc cells were mock-transfected or transfected with indicated plasmid, followed by subcutaneous inoculation into NSG mice. Tumor growth was then monitored over time using in vivo bioluminescent imaging. Data are expressed as mean ± standard deviation, n=5. TCS: trichosanthin; hHCC: human hepatocellular carcinoma; Fluc: firefly luciferase."

Figure 7

Intratumor delivery of TCS gene through rAAV3 vectors significantly inhibits growth of established hHCC tumor Huh7-Fluc cells were subcutaneously injected into NSG mice for 3 weeks, followed by intratumor injection of indicated rAAV3 vectors. Tumor growth was then monitored over time using in vivo bioluminescent imaging. Data are expressed as mean ± standard deviation, n=5. TCS: trichosanthin; rAAV3: recombinant adeno-associated virus serotype 3; hHCC: human hepatocellular carcinoma."

[1] Xu J, Yang Y . Traditional Chinese medicine in the Chinese health care system[J]. Health Policy, 2009,90(2-3):133-139
doi: 10.1016/j.healthpol.2008.09.003
[2] Qiu J . China plans to modernize traditional medicine[J]. Nature, 2007,446(7136):590-591
doi: 10.1038/446590a
[3] Tang JL, Liu BY, Ma KW . Traditional Chinese medicine[J]. Lancet, 2008,372(9654):1938-1940
doi: 10.1016/S0140-6736(08)61354-9
[4] Dong S, Su SB . Advances in mesenchymal stem cells combined with traditional Chinese medicine therapy for liver fibrosis[J]. J Integr Med, 2014,12(3):147-155
doi: 10.1016/S2095-4964(14)60022-4
[5] Song HY, Zhang L, Pan JL, Yang LL, Ji G . Bioactivity of five components of Chinese herbal formula Jiangzhi granules against hepatocellular steatosis[J]. J Integr Med, 2013,11(4):262-268
doi: 10.3736/jintegrmed2013034
[6] Zhai XF, Chen Z, Li B, Shen F, Fan J, Zhou WP, Yang YK, Xu J, Qin X, Li LQ, Ling CQ . Traditional herbal medicine in preventing recurrence after resection of small hepatocellular carcinoma: a multicenter randomized controlled trial[J]. J Integr Med, 2013,11(2):90-100
doi: 10.3736/jintegrmed2013021
[7] Ling CQ, Wang LN, Wang Y, Zhang YH, Yin ZF, Wang M, Ling C . The roles of traditional Chinese medicine in gene therapy[J]. J Integr Med, 2014,12(2):67-75
doi: 10.1016/S2095-4964(14)60019-4
[8] Ng TB, Chan WY, Yeung HW . Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants[J]. Gen Pharmacol, 1992,23(4):579-590
[9] Shaw PC, Chan WL, Yeung HW, Ng TB . Minireview: trichosanthin ― a protein with multiple pharmacological properties[J]. Life Sci, 1994,55(4):253-262
doi: 10.1016/0024-3205(94)00727-6
[10] Fang EF, Ng TB, Shaw PC, Wong RN . Recent progress in medicinal investigations on trichosanthin and other ribosome inactivating proteins from the plant genus Trichosanthes[J]. Curr Med Chem, 2011,18(28):4410-4417
doi: 10.2174/092986711797200499
[11] Sha O, Niu J, Ng TB, Cho EY, Fu X, Jiang W . Anti-tumor action of trichosanthin, a type 1 ribosome-inactivating protein, employed in traditional Chinese medicine: a mini review[J]. Cancer Chemother Pharmacol, 2013,71(6):1387-1393
doi: 10.1007/s00280-013-2096-y
[12] Fang EF, Zhang CZ, Zhang L, Wong JH, Chan YS, Pan WL, Dan XL, Yin CM, Cho CH, Ng TB . Trichosanthin inhibits breast cancer cell proliferation in both cell lines and nude mice by promotion of apoptosis[J]. PLoS One, 2012,7(9):e41592
doi: 10.1371/journal.pone.0041592
[13] Shaw PC, Yung MH, Zhu RH, Ho WK, Ng TB, Yeung HW . Cloning of trichosanthin cDNA and its expression in Escherichia coli[J]. Gene, 1991,97(2):267-272
doi: 10.1016/0378-1119(91)90061-F
[14] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ . Growth arrest and apoptosis of the human hepatocellular carcinoma cell line BEL-7402 induced by melittin[J]. Onkologie, 2006,29(8-9):367-371
[15] Zhang K, Xu J, Huang X, Wu L, Wen C, Hu Y, Su Y, Chen Y, Zhang Z . Trichosanthin down-regulated p210Bcr-Abl and enhanced imatinib-induced growth arrest in chronic myelogenous leukemia cell line K562[J]. Cancer Chemother Pharmacol, 2007,60(4):581-587
doi: 10.1007/s00280-007-0457-0
[16] Lu J, Yang H, Yu H, Gao W, Lai R, Liu J, Liang X . A novel serine protease inhibitor from Bungarus fasciatus venom[J]. Peptides, 2008,29(3):369-374
doi: 10.1016/j.peptides.2007.11.013
[17] Zhao Y, Cai X, Ye T, Huo J, Liu C, Zhang S, Cao P . Analgesic-antitumor peptide inhibits proliferation and migration of SHG-44 human malignant glioma cells[J]. J Cell Biochem, 2011,112(9):2424-2434
doi: 10.1002/jcb.v112.9
[18] Liu S, Yu M, He Y, Xiao L, Wang F, Song C, Sun S, Ling C, Xu Z . Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway[J]. Hepatology, 2008,47(6):1964-1973
doi: 10.1002/(ISSN)1527-3350
[19] Mingozzi F, High KA . Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges[J]. Nat Rev Genet, 2011,12(5):341-355
[20] Melchiorri D, Pani L, Gasparini P, Cossu G, Ancans J, Borg JJ, Drai C, Fiedor P, Flory E, Hudson I, Leufkens HG, Müller-Berghaus J, Narayanan G, Neugebauer B, Pokrotnieks J, Robert JL, Salmonson T, Schneider CK . Regulatory evaluation of Glybera in Europe ― two committees, one mission[J]. Nat Rev Drug Discov, 2013,12(9):719
doi: 10.1038/nrd3835-c1
[21] Cheng B, Ling C, Dai Y, Lu Y, Glushakova LG, Gee SW , McGoogan KE, Aslanidi GV, Park M, Stacpoole PW, Siemann D, Liu C, Srivastava A, Ling C.Development of optimized AAV3 serotype vectors: mechanism of high-efficiency transduction of human liver cancer cells[J]. Gene Ther, 2012,19(4):375-384
doi: 10.1038/gt.2011.105
[22] You H, Ding W, Dang H, Jiang Y , Rountree CB.c-Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma[J]. Hepatology, 2011,54(3):879-889
[23] Ling C, Lu Y, Kalsi JK, Jayandharan GR, Li B, Ma W, Cheng B, Gee SW , McGoogan KE, Govindasamy L, Zhong L, Agbandje-McKenna M, Srivastava A.Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3[J]. Hum Gene Ther, 2010,21(12):1741-1747
doi: 10.1089/hum.2010.075
[24] Lan T, Rao A, Haywood J, Davis CB, Han C, Garver E, Dawson PA . Interaction of macrolide antibiotics with intestinally expressed human and rat organic anion-transporting polypeptides[J]. Drug Metab Dispos, 2009,37(12):2375-2382
doi: 10.1124/dmd.109.028522
[25] Wang R, Kniazeva M, Han M . Peroxisome protein transportation affects metabolism of branched-chain fatty acids that critically impact growth and development of C. elegans[J]. PLoS One, 2013,8(9):e76270
doi: 10.1371/journal.pone.0076270
[26] Liu Y, Fang Y, Zhou Y, Zandi E, Lee CL, Joo KI, Wang P . Site-specific modification of adeno-associated viruses via a genetically engineered aldehyde tag[J]. Small, 2013,9(3):421-429
doi: 10.1002/smll.201201661
[27] Dai Y, Bae K, Siemann DW . Impact of hypoxia on the metastatic potential of human prostate cancer cells[J]. Int J Radiat Oncol Biol Phys, 2011,81(2):521-528
doi: 10.1016/j.ijrobp.2011.04.027
[28] Dai Y, Siemann DW . BMS-777607, a small-molecule met kinase inhibitor, suppresses hepatocyte growth factor-stimulated prostate cancer metastatic phenotype in vitro[J]. Mol Cancer Ther, 2010,9(6):1554-1561
doi: 10.1158/1535-7163.MCT-10-0359
[29] Gu Y, Lin S, Li JL, Nakagawa H, Chen Z, Jin B, Tian L, Ucar DA, Shen H, Lu J, Hochwald SN, Kaye FJ, Wu L . Altered LKB1/CREB-regulated transcription co-activator(CRTC) signaling axis promotes esophageal cancer cell migration and invasion[J]. Oncogene, 2011,31(4):469-479
[30] Liu B, Behura SK, Clem RJ, Schneemann A, Becnel J, Severson DW, Zhou L . P53-mediated rapid induction of apoptosis conveys resistance to viral infection in Drosophila melanogaster[J]. PLoS Pathog, 2013,9(2):e1003137
doi: 10.1371/journal.ppat.1003137
[31] Dai Y, Desano J, Qu Y, Tang W, Meng Y, Lawrence TS, Xu L . Natural IAP inhibitor Embelin enhances therapeutic efficacy of ionizing radiation in prostate cancer[J]. Am J Cancer Res, 2011,1(2):128-143
[32] Liu Y, Joo KI, Wang P . Endocytic processing of adeno-associated virus type 8 vectors for transduction of target cells[J]. Gene Ther, 2012,20(3):308-317
[33] Wang Y, Ling C, Song L, Wang L, Aslanidi GV, Tan M, Ling C, Srivastava A . Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation[J]. Hum Gene Ther Methods, 2012,23(4):225-233
doi: 10.1089/hgtb.2012.090
[34] Liu Y, Kim JY, Man J, Fang J, Siriwon N, Zhang L, Wang P . Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides[J]. Mol Ther Methods Clin Dev, 2014,1:12
doi: 10.1038/mtm.2013.12
[35] Lin S, Tian L, Shen H, Gu Y, Li JL, Chen Z, Sun X, You MJ, Wu L . DDX5 is a positive regulator of oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia[J]. Oncogene, 2012,32(40):4845-4853
[36] Ling CQ, Yue XQ, Ling C . Three advantages of using traditional Chinese medicine to prevent and treat tumor[J]. J Integr Med, 2014,12(4):331-335
doi: 10.1016/S2095-4964(14)60038-8
[37] Bosch MK, Nerbonne JM, Ornitz DM . Dual transgene expression in murine cerebellar purkinje neurons by viral transduction in vivo[J]. PLoS One, 2014,9(8):e104062
doi: 10.1371/journal.pone.0104062
[38] Li M, Li X, Li JC . Possible mechanisms of trichosanthin-induced apoptosis of tumor cells[J]. Anat Rec (Hoboken), 2010,293(6):986-992
doi: 10.1002/ar.21142
[39] Liu Y, Fang J, Kim YJ, Wong MK, Wang P . Codelivery of doxorubicin and paclitaxel by cross-linked multilamellar liposome enables synergistic antitumor activity[J]. Mol Pharm, 2014,11(5):1651-1661
doi: 10.1021/mp5000373
[40] Zhang Y, Zhao L, Li X, Wang Y, Yao J, Wang H, Li F, Li Z, Guo Q . V8, a newly synthetic flavonoid, induces apoptosis through ROS-mediated ER stress pathway in hepatocellular carcinoma[J]. Arch Toxicol, 2013,88(1):97-107
[1] Brody Slostad, Tejinder Khalsa, Kathleen Young, Hildalicia Guerra, Anjali Bhagra. A case-based approach to integrative medicine for cardiovascular disease prevention. Journal of Integrative Medicine, 2020, 18(2): 159-162.
[2] Ameena Yasmeen, Ghulamuddin Sofi, Kaleemullah Khan. Aamar-e-Advia (shelf-lives) of drugs in Unani system of medicine: A conceptual review. Journal of Integrative Medicine, 2020, 18(2): 114-124.
[3] Louisa Sylvia, Emerson West, Allyson M. Blackburn, Carina Gupta, Eric Bui, Tara Mahoney, Geraldine Duncan, Edward C. Wright, Simon Lejeune, Thomas J. Spencer. Acceptability of an adjunct equine-assisted activities and therapies program for veterans with posttraumatic stress disorder and/or traumatic brain injury. Journal of Integrative Medicine, 2020, 18(2): 169-173.
[4] Sherman Gu, Arthur Yin Fan. Controversial conclusions from two randomized controlled trials for acupuncture's effects on polycystic ovary syndrome or in vitro fertilization support. Journal of Integrative Medicine, 2020, 18(2): 89-91.
[5] Xue-qing Yu, Shu-guang Yang, Yang Xie, Jian-sheng Li. Traditional Chinese medicine in the treatment of idiopathic pulmonary fibrosis based on syndrome differentiation: Study protocol of exploratory trial. Journal of Integrative Medicine, 2020, 18(2): 163-168.
[6] Chang-quan Ling. Traditional Chinese medicine is a resource for drug discovery against 2019 novel coronavirus (SARS-CoV-2). Journal of Integrative Medicine, 2020, 18(2): 87-88.
[7] Sajjad Sadeghi, Farzaneh Ghaffari, Ghazaleh Heydarirad, Mehdi Alizadeh. Galen’s place in Avicenna’s The Canon of Medicine : Respect, confirmation and criticism. Journal of Integrative Medicine, 2020, 18(1): 21-25.
[8] Maryam Naeimia, Narjes Gorjib, Zahra Memarianib, Reihaneh Moeinib, Mohammad Kamalinejadc, Fatemeh Kolangid. Gastroprotective herbs for headache management in Persian medicine: A comprehensive review. Journal of Integrative Medicine, 2020, 18(1): 1-13.
[9] Mohadeseh Ostovar, Abolfazl Akbari, Mohammad Hossein Anbardar, Aida Iraji, Mohsen Salmanpour, Salar Hafez Ghoran, Mojtaba Heydari, Mesbah Shams. Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy. Journal of Integrative Medicine, 2020, 18(1): 59-67.
[10] Preecha Nootim, Nattiya Kapol, Waranee Bunchuailua, Panoopat Poompruek, Parankul Tungsukruthai . Current state of cancer patient care incorporating Thai traditional medicine in Thailand: A qualitative study. Journal of Integrative Medicine, 2020, 18(1): 41-45.
[11] Md. Anzar Alam, Mohd Aleemuddin Quamri, Ghulamuddin Sofi, Barkati Md.Tarique. Understanding hypothyroidism in Unani medicine. Journal of Integrative Medicine, 2019, 17(6): 387-391.
[12] Naghmeh Yazdi, Alireza Salehi, Mina Vojoud, Mohammad Hossein Sharifi, Ayda Hoseinkhani. Use of complementary and alternative medicine in pregnant women: a cross-sectional survey in the south of Iran. Journal of Integrative Medicine, 2019, 17(6): 392-395.
[13] Wen Qiang Lee, Jeremy Teoh, Pei Zheng Kenneth Lee, Zhi Xiong Gerard Low, Xueling Sim, Foong Fong Mary Chong, Norbert Ludwig Wagner. Factors influencing communication of traditional Chinese medicine use between patients and doctors: a multisite cross-sectional study. Journal of Integrative Medicine, 2019, 17(6): 396-403.
[14] Zienab Mahmoudpour, Javad Shokri, Mohammad Kamalinejad, Neda Meftah, Soraya Khafri, Seyyed Ali Mozaffarpur, Hoda Shirafkan. The efficacy of a Persian herbal formulation on functional bloating: A double-blind randomized controlled trial. Journal of Integrative Medicine, 2019, 17(5): 344-350.
[15] Claire L. Hoban, Roger W. Byard, Ian F. Musgrave. Analysis of spontaneous adverse drug reactions to echinacea, valerian, black cohosh and ginkgo in Australia from 2000 to 2015. Journal of Integrative Medicine, 2019, 17(5): 338-343.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. An explanation on "guiding principles of clinical research on mild cognitive impairment (protocol)". Journal of Chinese Integrative Medicine, 2008, 6(1): 15-21
[2] Jin-rong Fu. Establishment of multivariate diagnosis and treatment system of modern gynecology of traditional Chinese medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 22-24
[3] Yi-ting He, Qing-lin Zha, Jian-ping Yu, Yong Tan, Cheng Lu, Ai-ping Lv. Principal factor analysis of symptoms of rheumatoid arthritis and their correlations with efficacy of traditional Chinese medicine and Western medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 32-36
[4] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[5] Wei Zhang, Xiang-feng Lu, Xiao-mei Zhang, Jian-jun Wu, Liang-duo Jiang. A rat model of pulmonary fibrosis induced by infusing bleomycin quickly through tracheal intubation. Journal of Chinese Integrative Medicine, 2008, 6(1): 60-67
[6] Xi Lin, Jian-ping Liu. Tai chi for treating rheumatoid arthritis. Journal of Chinese Integrative Medicine, 2008, 6(1): 82
[7] Guo-hong Yuan, Xiao-jing Pang, He-chao Ma. Synergic effects of Danggui Buxue Decoction in reducing toxicity of cytoxan in tumor-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 83-88
[8] Li Zhou, Hong-xing Zhang, Ling-guang Liu, Wen-jun Wan. Effect of electro-acupuncture at Fenglong (GV 16) on nitric oxide and endothelin in rats with hyperlipidemia. Journal of Chinese Integrative Medicine, 2008, 6(1): 89-92
[9] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. Guiding principles of clinical research on mild cognitive impairment (protocol). Journal of Chinese Integrative Medicine, 2008, 6(1): 9-14
[10] Ning-qun Wang, Liang-duo Jiang, Zong-xing Li. Research progress in asthma-related quality of life. Journal of Chinese Integrative Medicine, 2008, 6(1): 93-97