How Does Cistanche Extract Reduce Inflammatory Hyperplasia
Mar 17, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Reduction of Inflammatory Hyperplasia in the Intestine in Colon Cancer-prone Mice by Water-extract of Cistanche deserticola
Yamin Jia,1,2 et al
Cistanche deserticola has commonly been used in traditional Chinese medicine to treat many health problems including irritable bowel syndrome or constipation. This study was designed to test the efficacy of a water extract of C. deserticola in the prevention of colorectal cancer in a mouse model. Polysaccharide-rich water extract of C. deserticola was prepared by boiling its stem powder in distilled water. Tgfb1Rag2 null mice were used as an experimental model. Here we showed that feeding of water extract of C. deserticola significantly reduced the number of mucosal hyperplasia and intestinal Helicobacter infection in mice. This beneficial effect correlated with significant stimulation of the immune system, evidenced by the enlargement of the spleens with an increased number of splenic macrophage and natural killer cells, and with more potent cytotoxicity of splenocytes. In vitro water extract of C. deserticola enhanced the cytotoxicity of naïve splenocytes against a human colon cancer cell line, and in macrophage cultures up-regulated nitric oxide synthase II expression and stimulated phagocytosis. In conclusion, our data indicate that oral administration of C. deserticola extract reduces inflammatory hyperplastic polyps and helicobacter infection in mice by its immune-stimulatory activity, suggesting that C. deserticola extract may have the potential in preventing intestinal inflammation disorders including colorectal cancer. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: herbal immunomodulation; Cistanche extract; polysaccharides; helicobacter; inflammatory hyperplasia; colorectal cancer.
Cistanche extract can reduce Inflammatory hyperplasia
INTRODUCTION
Colorectal cancer is the third most common cancer in men and the second in women worldwide and ranks the fourth most common cause of death from cancer (8% of all cancer death; Ferlay et al., 2010). Epidemiological studies suggest that many risk factors associated with this disease, including genetic (e.g. family history of colon cancer) (Strate and Syngal, 2005) and dietary factors (e.g. red meat and low –fiber; Chao et al., 2005; Park et al., 2005), and inflammatory bowel diseases (IBD; Eaden et al., 2001; von Roon et al., 2007; Lukas, 2010). To date, despite the many new cytotoxic drugs and improved surgical and radiotherapeutic techniques that are used for the treatment of this disease (Cunningham et al., 2010), a large proportion of patients with colorectal cancer still remain incurable; about 608000 deaths from this disease are estimated worldwide in 2008 (Ferlay et al., 2010). Hence, novel therapies are needed, and one candidate is immunotherapy. Various immunotherapeutic approaches have been evaluated for the treatment of colorectal cancer, and results so far are encouraging (Burgdorf et al., 2009). Herbal polysaccharides have been demonstrated to have specific immunoregulatory activities (Jiang et al., 2010), suggesting that these herbal products could be used as a specific immunostimulatory adjuvant against colorectal cancer.
Cistanche deserticola Ma, known as Rou Cong Rong in Chinese herb medicine, has been included in herbal formulas for many health problems, including impotence, morbid leucorrhea, and irritable bowel syndrome/constipation (Jiang and Tu, 2009). Experimental studies have revealed different biological activities by different extracts of C. deserticola; the phenylethanoid glycosides extract of this herbal has antifatigue activity in mice (Cai et al., 2010), and analgesic and anti-inflammatory effects in rodents (Lin et al., 2002), while its polysaccharides stimulate T- and B-lymphocyte proliferation in lymphocyte cultures (Wu et al., 2005; Dong et al., 2007). However, the efficacy of polysaccharides extract of C. deserticola in the stimulation of immune response in vivo has not been examined yet.
Rag2-/- mice lack mature T- and B-lymphocytes, and on the mixed background (~97% 129 S6 and ~3% CF-1) spontaneously develop inflammation-associated hyperplasia in the intestine (Engle et al., 1999). Genetic introduction of the Tgfb1 (transforming growth factor b1 (TGF-b1) gene) null allele to this strain of mice results in further progression of hyperplasia to adenoma and carcinoma (Engle et al., 2002), and the pathogenic microflora-dependent colitis is required for the development of of the colon cancer (Engle et al., 2002), suggesting that this strain of mice provides an ideal model for the examination of a potential therapy in the treatment of inflammation/IBD-induced colorectal cancer. In this study, we demonstrate for the first time the efficacy of polysaccharide-rich water extract of C. deserticola in the reduction of inflammatory hyperplasia in this model.
MATERIALS AND METHODS
Polysaccharide-rich water extract of C. deserticola preparation.
Herbal C. deserticola was harvested by Dr. Y. Guo (China Agricultural University) in the autonomous region of Inner Mongolia, China. The powder of whole dried stems (300 g) was boiled in double-distilled water twice: first for 1 h in 500 mL of water, followed by a collection of the supernatant, then for 45 min in 400 mL of water. The supernatants were filtrated through a Whatman filter paper (medium fast) after each time of boiling, and pooled together. The extract solution was dried by lyophilization and was kept at 80C. The final product was about 10 g (yield: ~3%).
Mice and cell cultures. TGF-b1 deficient mice (Tgfb1+/- Rag2-/-) on the mixed background (~97% 129 S6 and ~3% CF-1) were received from a breeding colony maintained under pathogen-free conditions in the animal facility at the Jack Bell Research Centre (Vancouver, BC). These mice originated from the Mouse Models of Human Cancers Consortium (MMHCC) Repository (National Cancer Institute at Frederick, MD, USA). C57BL/6 J (B6) mice (male, 8–10 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All the animals for the experiments were kept under conventional housing and diet (5001 Rodent Diet, LabDiet) after weaning at 4 weeks old, and herbal treatment and sample collection were performed in accordance with the Canadian Council on Animal Care guidelines under
protocols approved by the Animal Use Subcommittee at the University of British Columbia.
Both the SW480 human colon adenocarcinoma cell line and the RAW 246.7 mouse leukemic macrophage cell line was grown in Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (complete DMEM medium) at 37-C in a humidified 5% CO2/95% air incubator. Splenocytes were isolated by gently smashing spleen fragments in phosphate-buffered saline (PBS), followed by removal of erythrocytes using lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA, pH 6.8). After washing with PBS again, splenocytes were suspended in RPMI 1640 medium supplemented with 10% FBS (complete RPMI medium).
Cistanche extract can reduce Inflammatory hyperplasia
Extract treatment.
The herbal extract, termed Cistanche extract in this study, was finally prepared by reconstitution of dried powder with sterilized double-distilled water and contained 2–3% (w/w) of phenylethanoids, 65–70% (w/w) of polysaccharides, and 0.6–1% (w/w) of protein-based on the following assays: the number of phenylethanoids was determined by absorbance at 332 nm using a previously described spectrophotometry (Ouyang et al., 2005); the content of polysaccharides was measured using a standard phenol sulphuric acid method (Dubois et al., 1956) after ethanol precipitation; and protein was determined by Bio-Rad protein assay following the manufacturer's protocol (Bio-Rad Lab, Hercules, CA, USA). The Cistanche extract dosage was 0.4 g/kg/day based on our pilot study. The average water intake per adult mouse per day was approximately 4 mL, and if 1 mL of drinking water contained 3 mg of Cistanche extract, each mouse received oral administration of 12 mg of the extract per day. Based on the fact that the average body weight of an adult mouse was 30 g, the drinking water containing 3 mg/mL of Cistanche extract gave a dosage of 0.4 g/kg/day to the mice. The mice were treated by giving drinking water ad labium containing 3 mg/mL of Cistanche extract that was replenished at least once every two days, from the age of two months for three months in a conventional room of the animal care facility.
Histological grading of colorectal cancer.
At the end of the three-month treatment, both small and large intestinal tissues were harvested and opened longitudinally, followed by gently flushing the contents out with PBS. A total of six pieces (each 2 cm long) of intestinal tissue from each mouse was collected: three from the small intestine at proximal, middle, and distal locations; two from the large intestine at proximal and distal locations, and one from the caecum. All tissues were fixed in 10% formalin and embedded in paraffin. Each section was stained with hematoxylin and eosin (HE) for histological grading of colorectal cancer. The disease grading was performed according to the pathological characters of hyperplasia, adenoma, or carcinoma within each sample, as previously described (Engle et al., 1999) in a blind test fashion. The total number of each pathological change (hyperplasia, adenoma, and carcinoma) was counted in two sections of the intestinal tissue for each mouse.
Cistanche extract can reduce Inflammatory hyperplasia
Detection of Helicobacter hepaticus (H. hepaticus) in mouse faeces.
To examine the effect of Cistanche extract on the growth of pathological microflora in the gut, H. hepaticus in the faeces was determined by polymerase chain reaction (PCR) with species-specific 16S rDNA primers as described previously (Shames et al., 1995). Approximately 200 mg of fresh faeces was dissolved in 1 μL of PBS and incubated at 50℃ for 30 min, followed by centrifugation at 8000 rpm for 10 min. The supernatant (200 mL) was collected and mixed with an equal amount of proteinase K solution containing 5% (v/v) of 18.7 mg/mL proteinase K solution (Fermentas Canada, Burlington, ON, Canada) in a digest buffer (50 μM Tris–HCl, pH 8.0, 0.1 M EDTA, 0.5% Sodium dodecyl sulfate), and incubated at 70 C for 30 min. The DNA in the resultant solution was extracted using the QIAprep Miniprep kit following the manufacturer's protocol (QIAGEN, Mississauga, ON, Canada).
Polymerase chain reaction amplification of bacterial DNA was performed in a mixture containing 0.75 μL of 10 PCR buffer, 0.5μL of 50 mM MgCl2, 0.5 μL of each primer (sense: 5'-GCA TTT GAA ACT GTT ACT CTG; anti-sense: 5'-GGG GAGCUUGAAAACAG, 100 mM each), 0.5 μL of Taq polymerase, 18.75 μL of nuclease-free H2O and 1μL of unquantitated faeces DNA solution. The PCR mixture was heated at 94 ℃ for 5 min, followed by 40 cycles of denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1.25 min, and extension at 72℃ for 1.5 min. The PCR was ended by an elongation step of 10 min at 72 ℃. The resultant PCR product (25 μL) was subjected to electrophoresis on a 1% agarose gel in Tris-acetate-EDTA (TAE) buffer containing 0.5 mg/mL of ethidium bromide and visualized under UV light.
Determination of macrophages and natural killer (NK) cells.
The percentages of macrophages and NK cells in splenocyte suspension were determined by fluorescence-activated cell sorting (FACS) analysis. One million splenocytes in FACS buffer were incubated with appropriate antibody combination for different phenotypes of cells in ice: anti-CD3-fluorescein isothiocyanate (FITC) and anti-CD49b-phycoerythrin (PE) (BD Biosciences, Mississauga, ON, Canada) for CD3CD49b+ NK cells, and rat anti-mouse F4/80+ as a primary antibody and anti-rat IgG-FITC as a secondary antibody (eBioscience, San Diego, CA, USA) for F4/80+ macrophages. The intensity of fluorescence for each type of cell was measured using flow cytometry and analyzed by comparison with background controls using CELLQUEST software (BD Biosciences).
Calcein-acetoxymethyl (calcein-AM) cytotoxicity assay.
The lytic activity of effector splenocytes against target SW480 cells was evaluated by calcein-AM cytotoxicity assay using a co-culture system as described previously (Neri et al., 2001). In brief, SW480 cells were labeled with fluorescent dye calcein-AM as follows: 1 x106 cells/mL of SW480 cells in complete DMEM medium were incubated with 15 μM calcein-AM at 37℃ with occasional shaking. After 30 min of incubation, cells were extensively washed with a complete DMEM medium to remove the extracellular dye. The fluorescent dye-labeled SW480 cells in complete DMEM medium (0.2 x 106 cells/0.25 mL/well) were seeded in 24-well plates. Based on the different ratios (12:1, 6:1, 3:1, and 1.5:1) of E (effector): T (target), splenocytes in 0.25 mL of complete RPMI medium per well were added to the wells with target cells in triplicate. Co-cultures were incubated at 37 ℃ in 5% of CO2 for 24 hr. Supernatants were collected after the plate was spun at 2000 rpm for 5 min. Calcein-AM release to the supernatant was determined by using a microplate spectrofluorimeter (Fluoroskan Ascent FL, Thermo Labsystems) at an emission of 527 nm under excitation at 485 nm. The target cells, incubated with a mixture of 0.25 mL of complete DMEM medium and 0.25 mL of complete RPMI medium, were used as background or spontaneous release controls, and in the presence of 2%, Triton X-100 was used as maximum release controls. The cytotoxicity of splenocytes was calculated as follows:
Nitric oxide (NO) measurement. Nitric oxide secreted from cells was rapidly oxidized to nitrite in the culture medium. Therefore, nitrate concentrations in the medium were determined using the Griess method as a measurement of NO production. RAW264.7 cells (0.25 x 106
cells/well in 0.5 mL of complete DMEM medium) in 24-well plates were stimulated with Cistanche extract (50–200 mg/mL) and lipopolysaccharide (LPS) (10 ng/mL) as a positive control. After 24 hr of treatment, 50 mL of culture supernatant were first mixed with 50 mL of 1%
sulphanilamide in 5% phosphoric acid in 96-well plates in triplicate, and were incubated for 10 min, then 50 mL of 0.1% naphthyl ethylene diamine dihydrochloride in distilled water were added per well. The absorbance was read at 550 nm after incubation for 10 min, and the level of NO/nitrite was calculated using a standard curve with known sodium nitrite concentrations.
Cistanche extract can reduce Inflammatory hyperplasia
Western blot.
Cellular levels of nitric oxide synthase II (NOS 2 or iNOS) were examined by Western blot as described previously (Du et al., 2006). Briefly, protein samples (approximately 100 mg/sample) were fractionated by 7% SDS-PAGE and were transferred onto the nitrocellulose membrane. The NOS II protein bands were identified with primary rabbit polyclonal anti-NOS II antibody (N-20) (1:500 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA) and secondary goat anti-rabbit IgG antibody (1:10000 dilution; Vector Laboratory, Burlingame, CA, USA). The NOS II protein–antibody complex was visualized by an enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech, Buckinghamshire, England). Blots were reprobed using anti-b-actin IgG (Sigma-Aldrich Canada, Oakville, ON, Canada) for confirmation of loaded protein in each sample.
Phagocytosis assay. Phagocytosis was measured by the amount of fluorescence-labeled latex beads (SigmaAldrich Canada, L-3030, carboxylate-modified, average size 2 mm) engulfed by RAW264.7 cells as described previously (Wu et al., 2007). In brief, RAW264.7 cells (0.1 106 cells/0.5 mL/well) in complete DMEM medium were seeded in 24-well plates and stimulated with 100 mg/mL of Cistanche extract or complete DMEM medium only as control. After overnight stimulation, 2.5 mL of fluorescent latex beads per well were added to the macrophage cultures and incubated for 2 hr. For flow cytometry analysis, cells were detached by pipetting after washing in PBS, and the intensity of the fluorescence of phagocytic cells was measured by a flow cytometer and analyzed by comparison with background controls (no beads) using CELLQUEST software (BD Biosciences).
Statistical analysis.
Data were presented as mean+-standard deviation (SD). Analysis of variance (ANOVA) or Student's t-test was performed as appropriate for comparing the data between groups. A p-value of < 0.05 was considered significant.
RESULTS
Treatment with Cistanche extract reduces the frequency of hyperplasia and H. hepaticus infection of the intestine During the period of treatment (3 months), the body weight and drinking water intake were monitored once every 2 days in mice of the extract-treated group versus the vehicle-treated group, no significant difference was noted (data not shown). Histological analysis, however, showed that oral administration of Cistanche extracts significantly reduced the number of inflammatory hyperplasia in the intestine (Fig. 1), evidenced by the fact that mice receiving water with extract had fewer hyperplasia (5.00 +-2.83/mouse, n = 15) than those drinking plain water or vehicle (7.53 +-3.09/mouse, n = 15) (extract vs. vehicle: p = 0.0133, t-test). The difference in the number of adenoma and carcinoma between extract- and vehicle-treated mice was not statistically significant because only a few mice had these stages of pathological changes (data not shown).
Figure 1. Reduction of the incidence of hyperplasia by treatment with water extract of C. deserticola (Cistanche extract). Tgfb1+/-Rag2-/- mice were fed with Cistanche extract in drinking water (Extract group) compared with those with plain drinking water only (Vehicle group). Cancerous lesions in the intestine were examined by histology with an H&E stain. (A) A typical image of the healthy tissue section. (B) A typical image of hyperplasia in the section, indicated by an arrow. (C) The number of hyperplasia in the intestine of each animal was counted by histology (p = 0.0133, extract versus vehicle, n = 15). This figure is available in color online at wileyonlinelibrary.com/journal/ptr
Gut microbial content, particularly H. hepaticus infection, has been demonstrated to be required for the development of all stages of colon cancer including hyperplasia in Rag2-/- mice (Engle et al., 2002; Erdman et al., 2003). To examine if the treatment with Cistanche extracts reduced pathological microflora in the gut, the presence of H. hepaticus in the faeces from the mice treated with Cistanche extract was determined by PCR as compared with vehicle controls. In each experiment, three mice per cage were treated with Cistanche extract or vehicle. The stool samples were collected at the end of a 14-h overnight period after the cage was cleaned. As shown in Fig. 2, H. hepaticus 16S rDNA in the faeces was efficiently detected by the PCR technique. In three separate experiments (Table 1), only 20–40% of the samples from Cistanche-extract-treated mice were detected as PCR positive, whereas 60–80% from vehicle controls were positive (extract vs. vehicle: p = 0.0189, t-test, n = 3), suggesting that treatment with Cistanche extract reduced intestinal H. hepaticus infection in mice.
Treatment with Cistanche extract increases the number of splenic macrophages and NK cells
The immune response plays a key role in eliminating helicobacter infection. To examine if Cistanche extract treatment affected the immune system in these colon cancer-prone mice, the size of the spleens (12 mice in each group), and the number of splenocytes (six mice randomly selected from each group) was determined at the end of treatment. The spleens of 12 mice in each group were weighed, the other three mice in the initial experiment were not included. As illustrated in Table 2, Cistanche-extract-treated mice (0.055+-0.022 g/spleen) were heavier than in the vehicle group (0.038+-0.01 g/spleen; extract vs. vehicle: p=0.04, t-test). These data were further supported by an increase in the number of splenocytes in mice treated with Cistanche extract, indicated by 7.095+-1.353 (x106 cells/spleen) compared with 2.941+-1.492 (x106 cells/spleen) in-vehicle control mice (extract vs. vehicle: p=0.002, t-test).
To further evaluate whether Cistanche extract treatment affected a particular subtype of splenocytes in mice, the number of splenic NK cells (CD3-CD49b+ cells) and macrophages (FT/80+ cells) was counted by FACS analysis. The percentage of CD3-CD49b+ cells in the splenocytes of Cistanche-extract-treated mice (32.06+-2.13%) was not significantly different from that in vehicle-treated mice (28.47+-2.74%; extract vs. vehicle: p=0.3741, t-test, n=6), but the total number of NK cells in the spleen of Cistanche-extract-treated mice (2.22+- 0.44x106/spleen) was higher than that in-vehicle control group (0.83 +-0.17x106/spleen; extract vs. vehicle: p=0.01, t-test, n=6; Fig. 3B). Similar results were seen in the examination of splenic FT/80+cells; as in the splenic NK cell population, the difference in the percentage of splenic FT/80+ cells between extract-treated mice (35.25+-7.28%) and vehicle control mice (31.74+-5.07%) was not significant (extract vs. vehicle: p=0.407, n=6), but the total number of splenic macrophages in the extract-treated group (2.439+-0.452x106 cells/spleen) was higher than that in control groups (1.114+-0.685x106 cells/spleen; extract vs. vehicle: p=0.001, t-test, n=6; Fig. 3D). Taken together, these data suggest that the treatment with Cistanche extract may increase the total number, but not percentage or proportion, of subtypes of effector splenocytes, NK cells, and macrophages in the spleens.
Figure 3. Increase in the number of splenic NK cells and macrophages by the treatment with Cistanche extract. NK cells or macrophages in splenocyte suspension were determined by FACS analysis with a combination stain of FITC-anti-CD3e antibody and APC-anti-CD49b/Pan-NK cell marker (for NK cells) or with a single stain of FITC-anti-FT/80 (for macrophages). (A) A representative graph of the percentage of splenic NK cells (CD3-CD49b+) in the splenocytes of mice treated with Cistanche extract versus vehicle. (B) The total number of NK cells per spleen. Data are presented as mean+-SD in each group (p= 0.01, n= 6). (C) A representative graph of the percentage of splenic macrophage (FT/80+) in the splenocytes of mice treated with Cistanche extract versus vehicle. (D) The total number of macrophages per spleen. Data are presented as mean+-SD in each group (p= 0.001, n=6).
Treatment with Cistanche extract stimulates splenocyte cytotoxicity in vivo and in vitro
The immune response depended on not only the number of splenocytes but also their function, such as cytotoxicity. The effect of Cistanche extract treatment on the function of splenocytes (i.e. cytotoxicity) was examined in co-cultures with SW480 cells. As shown in Fig. 4A, the cytotoxicity of splenocytes from mice treated with Cistanche extract was higher than that of cells from vehicle-treated mice, indicated by 39.38+-2.64% of calcein-AM release in the co-cultures of extract-treated splenocytes with SW480 cells at a 6:1 ratio, 51.11 +-2.6% at a 3:1 ratio and 57.33+-2.53% at a 1.5:1 ratio, as compared with 27.75- 0.18% in the co-cultures of vehicle-treated splenocytes with SW480 cells at a 6:1 ratio, 40.97 =-2.85% at a 3:1 ratio and 45.16+-0.18% at a 1.5:1 ratio (extract vs. vehicle: p < 0.0001, two-way ANOVA). To further confirm the stimulatory effect of Cistanche extract on splenocyte cytotoxicity, the cytotoxicity of Cistanche-extract-treated naïve splenocytes from B6 mice, containing functional T and B cells, was examined in the co-cultures with SW480 cells. As shown in Fig. 4B, addition of Cistanche extract (100 mg/mL) significantly increased the calcein-AM release in the co-cultures in a splenocyte dependent manner: 57.64+-3.41% at a 12:1 ratio, 52.25+-0.77% at a 6:1 ratio and 45.72+-2.43% at a 3:1 ratio, as compared with 14.36+-0.05% at a 12:1 ratio, 20.67+-2.03% at a 6:1 ratio and 23.1+-3.73% at a 3:1 ratio in unstimulated co-cultures (extract vs. vehicle: p < 0.0001, two-way ANOVA). These results indicate that Cistanche extract stimulates the lytic activity of splenocytes. The reason why less cytotoxicity was seen when more effector splenocytes were added is unknown. One possibility was that the level of calcein-AM release in this co-culture system might not exactly reflect the target cell death because the calcein-AM could be reabsorbed by effector splenocytes after a long period of incubation.
Cistanche extract activates macrophages
Activation of macrophages is one of the critical immune responses against infection and tumor development (Mantovani et al., 2002, 2008). To further reveal insight into the mechanisms by which Cistanche extract treatment reduced intestinal hyperplasia and microbial content, the effect of Cistanche extract on the NO production and phagocytosis of macrophages, RAW264.7 cells, was examined. As shown in Fig. 5A, protein expression of NOS II was up-regulated in cultures following incubation with Cistanche extract. These results were further supported by the increase in NO production in Cistanche extract-stimulated cultures (Fig. 5B), evidenced by 3.88+-0.13 mM of nitrite in 50 mg/mL of Cistanche extract stimulated RAW264.7 cell cultures compared with 0.11+-0.12 mM of nitrite in unstimulated control cultures (one-way ANOVA, p<0.0001).
Phagocytosis is a cellular process of engulfing solid particles, such as bacteria. As shown in Fig. 5C and D, the addition of Cistanche extracts stimulated macrophage phagocytosis, as indicated by more phagocytic cells (9.8+-0.1%) being found in Cistanche-extract-stimulated macrophage cultures compared with those (5.2+-0.4%) in vehicle-stimulated cultures (extract vs. vehicle: p=0.0007, t-test, n=3) after a 2 h incubation. These data suggest that Cistanche extract may directly activate macrophages by up-regulation of local NO production and stimulation of macrophage phagocytosis in the gut of mice.
DISCUSSION
The present study demonstrates for the first time that oral administration of Cistanche extract significantly reduces the frequency of hyperplasia and H. hepaticus infection in the gut of colon-cancer-prone mice. The beneficial effect of Cistanche extract treatment is associated with an increase in the number and cytotoxicity of splenic NK cells and macrophages. In vitro addition of Cistanche extract stimulates NO production and phagocytosis in macrophages. TGF-b1 deficient Rag2-/-mice have been demonstrated to develop colon cancer (hyperplasia, adenoma, or adenocarcinoma) from 3 to 6 months of age, and it has been demonstrated that inflammatory foci are always associated with these tumourigenic lesions (Engle et al., 1999). Further studies have demonstrated that in these mice colon cancer development is required for the presence of the infection of pathogenic microbial flora because colon cancer is completely eliminated by housing mice in germ-free conditions (Engle et al., 2002), and H. hepaticus infection also induces colitis and large bowel carcinoma in Rag2-/-mice (Erdman et al., 2003). Similarly, in humans, microbial status and host immunity are the key factors for the progression of IBD (Fiocchi, 1998), and gastric carcinoma has been reported to be associated with H. pylori-induced chronic infection or inflammation (Parsonnet et al., 1991; El-Omar and Malfertheiner, 2001). The findings from these studies suggest that gastrointestinal infection may play a pivotal role in the development of colon cancer. Thus, targeting gastrointestinal infection may become a therapeutic strategy for the prevention or treatment of colon cancer as well as IBD. Our study demonstrates for the first time that oral administration of Cistanche extract reduces the frequency of inflammatory hyperplasia in the gut, and the number of H. hepaticus in the faeces in TGF-b1 deficient mice. The beneficial effect of Cistanche extract may be due to its activation of macrophages, as seen by an increase in both NO production and phagocytosis following treatment with Cistanche extract (Fig. 5), which may limit the intestinal pathogenic microflora including helicobacter (Mantovani et al., 2002, 2008). As a consequence, the lesion of inflammatory hyperplasia was reduced.
A variety of bioactive compounds have been identified in the extracts of C. deserticola including phenylethanoid glycosides and polysaccharides (Jiang and Tu, 2009). The phenylethanoid glycosides are cytoprotective, indicated by the fact that these compounds prevent cell death in cultured hepatocytes and cerebellar granule neurons (Xiong et al., 1998; Pu et al., 2003). While polysaccharides stimulate mitogen-induced T- and B-lymphocyte proliferation (Wu and Tu, 2005; Wu et al., 2005; Dong et al., 2007). It has been well documented that herbal polysaccharides modulate the immune response (Jiang et al., 2010). Cistanche extract contains 2–3% of phenylethanoids and 65–70% of polysaccharides, suggesting that the immunomodulation effect of Cistanche extract treatment might be due to the immunoregulatory activity of polysaccharides, the majority of bioactive substances in Cistanche extract. However, its target immune cells (e.g. macrophages or NK cells, or both) and molecular pathways require further investigation. In addition, a recent study demonstrates that administration of herbal phenylethanoid acetonide, one of the bioactive phenylethanoids in the extract of C. deserticola (Xiong et al., 1996; Jiang and Tu, 2009), has a beneficial effect on the reduction of dextran sulfate-sodium-induced colitis in mice (Hausmann et al., 2007), evidencing that the phenylethanoids (2–3%) in the Cistanche extract may contribute at least in part to its efficacy in the decrease in inflammatory hyperplasia, which also requires evaluation in the future.
In conclusion, colorectal cancer is one of the most prevalent cancers worldwide and is preventable in the majority of cases. The use of non-toxic herbal extracts may offer an effective strategy for the prevention of colorectal cancer development, and may also be applied as a chemo-adjuvant, which can benefit the enhancement of chemotherapeutic anticancer efficacy, and/or the reduction of the chemotherapeutic side effects. The data presented in this study clearly demonstrate that the treatment with water extract of C. deserticola reduces the hyperplasia and H. hepaticus infection in the gut of colon-cancer-prone mice. Helicobacter infection or bowel inflammation hyperplasia is a risk factor for colorectal cancer development. Our current study indicates that the use of this herb may have the potential for the prevention and treatment of colorectal cancer, particularly for those individuals who have IBD.
Acknowledgment
YM was supported by a graduate student scholarship from China Scholarship Council.
Conflict of Interest
The authors have no conflict of interest.
Cistanche extract can reduce Inflammatory hyperplasia
From: 'Reduction of Inflammatory Hyperplasia in the Intestine in Colon Cancer-prone Mice by Water-extract of Cistanche deserticola ' by Yamin Jia,1,2 et al
---Copyright © 2011 John Wiley & Sons, Ltd. Phytother. Res. 26: 812–819 (2012) DOI: 10.1002/ptr.3637
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