Abstract Objective To investigate the Weining granule of precancerous lesions of therapeutic effects and possible mechanisms. Methods 60 rats were randomly divided into blank group, model group, the retinoic acid group, Weining particles low dose group, middle dose group of Weining granule, Weining granule high dose group. General rats before and after treatment, gastric mucosa histopathology, telomerase activity, cell proliferation index (PI), apoptotic index (AI) changes. Results the Weining particles each dose group activity, diet and general situation improves, intestinal metaplasia (IM), atypical hyperplasia (ATP), the telomerase-positive cases significantly lower than the model group (P <0.05 or P <0.01) ; the Weining particles in each dose group cell proliferation index (PI), compared with the model group was significantly lower (P <0.01), the apoptosis index (AI) was significantly higher (P <0.05). Conclusion of Weining particles of precancerous lesions have a therapeutic effect, the mechanism may promote precancerous lesions of apoptosis and inhibition of telomerase activity, inhibited proliferation.

Key words gastric precancerous lesions of Weining granule

    Gastric cancer accounted for in the worldwide the various malignancies death 2, also ranks second in China and no significant downward trend [1]. A large number of studies have shown that the incidence of gastric cancer is a long process of evolution. Normal gastric mucosa → the superficial gastritis → atrophic gastritis → intestinal metaplasia → dysplasia → incidence of gastric cancer (intestinal) mode has been generally accepted. Intervene in the development process occurs in gastric carcinogenesis, and to be effective in reducing the incidence of gastric cancer.

    1 Materials and methods

    1.1 Animals

    60 healthy male Wistar rats, weight 80 ~ 100g, provided by the Experimental Animal Center of Guangxi Medical University.

    1.2 Drugs

    Weining granule century music: Guangxi College of Traditional Chinese Medicine Pharmaceutical Factory, product lot number 20050806; retinoic acid capsule: Chongqing Winbond Pharmaceutical Co., Ltd., batch number 20040607.

    1.3 Instruments and reagents

    ALLEGRA  64R centrifuge at low temperature: Beckman; the GH802  type single-person level clean bench; Tianjin biological clean plant products; in stillwellite microplate reader type III: Austria TECAN company’s products. Telomerase activity detection kit: Sino-American Biotechnology Company; proliferation assay kit: Wuhan Boster Biological Engineering Co., Ltd.; apoptosis detection kit: Wuhan Boster Biological Engineering Co., Ltd.; N  A  N  nitro  nitrosoguanidine (MNNG): the German FluKa products.

    1.4 grouping method

    60 Wistar rats were randomly divided into blank group, model group, the retinoic acid group, Weining particles low dose group, middle dose group of Weining granule, Weining granule high dose group (hereinafter referred to as low, medium and high dose groups), each Group 10. The adaptive feeding for one week, in addition to the blank rats reared normal, the other five groups of rats modeling.

    1.5 modeling methods

    Reference to land people [2] method and make improvements. Specific methods are as follows: daily free drink 100μg/ml MNNG solution (water bottles dark paint blackened), eating pure rat pellet feed containing 0.03% ranitidine morning with 56 ℃ 15% saline 10ml/kg gavage, fasting, and the afternoon 10ml/kg gavage with 40% alcohol. Week rat tail with pliers, so keep angered battle state continued for 30 min, the above method continuous modeling for 24 weeks.

    1.6 The method of administration

    Weining particles high in the low-dose group were equivalent dose of 4:2:1 (rat and human body surface area ratio calculation). Equivalent dose formula: rat equivalent dose = man-day dosage × 0.018 × 5. Dubbed in aqueous solution, the high-dose group crude drug content 2.088g/ml, the dose group crude drug content 1.044g/ml, 0.522g/ml crude drug content of the low-dose group. Retinoic acid group: rats dose 2 times the amount of the equivalent dose calculation, dubbed in aqueous solution, crude drug content 0.0762g/ml. Blank and model groups: 0.9% saline. The volume of each group according 10ml/kg body weight by gavage. 10 weeks for a course of treatment, a treatment courses. The rats were sacrificed 24 h after treatment, to detect relevant indicators.

    1.7 indexes

    1.7.1 In general

    Observed in each group diet, mental state, physical activities, Mao Guangze degrees, stool and survival.

    1.7.2 pathologic changes of gastric mucosa

    Open the stomach along the greater curvature, visual observation of the gastric mucosa of color, folds change, the stomach wall thickness, mucosal surface hyperplastic nodules, erosions and ulcers, etc..

    1.7.3 gastric mucosal histological changes

    The observed mucosal histopathological changes count each group superficial gastritis (CSG), atrophic gastritis (CAG), intestinal metaplasia (IM), the number of cases of atypical hyperplasia (ATP).

    1.7.4 Determination of telomerase activity

    Single-tube method step telomeric repeat amplification process. Gastric mucosa, 50μl of lysate was centrifuged, washed, added to ice bath, and the supernatant after centrifugation 2μl TRAP reaction, plus 45μl reaction mixture, liquid paraffin sealing set after the 25 ° C water bath for 30min, 94 ° C 5min inactivated telomerase, and then line PCR cycle the PCR end of the loop, the hybridization reaction, washing, secondary hybridization reaction, plate washer, adding color Yexian color, and finally placed in a microplate reader judgment result, wavelength 450nm read the OD value of specimen OD value greater than 2.1 times the negative control OD value sentenced for telomerase activity was positive.

    The 1.7.5 cell proliferation index (PI) Determination

    Using PCNA law. Counting five fields of view the number of apoptotic cells and cancer cells of the total number of steps in accordance with the requirements of the kit, and the average calculating PI. PI = PCNA-positive cells / total number of cells × 100%.

    1.7.6 apoptosis index (AI) Determination

    DNA ends by TdT-mediated in situ labeling staining apoptosis. Counting five fields of view the number of apoptotic cells and cancer cells of the total number of steps in accordance with the requirements of the kit, and the average value calculated AI. AI = the number of TUNEL-positive cells / total number of cells × 100%.

    1.8 statistical methods

    All data were statistically analyzed using SPSS13.0 count data using χ2 test, analysis of variance was used to compare the measurement data group.

    2 Results

    2.1 rats in each group general changes

    Normal group is always shaped fat body fat, eat more, activities sensitive Maoguang Ze; the model group emaciation, reduced activity, hair dull, less appetite, loose stool; the Weining particles is low, medium and high dose treatment group performance improved to varying degrees, food intake and increased activity, weight gain, stool became normal; retinoic acid rats appetite increase, but less shiny coat, weight increased stool forming.

    The 2.2 rat gastric mucosa general pathological changes

    Normal group, the gastric mucosa was pink, shiny, and covered more smooth surface mucus, mucosal folds, to the rules, and good elasticity of the stomach wall, no hyperplastic nodules. Model 24 weeks rat gastric parietal stretch weakened, thinning of the mucous layer of pale or dark purple color, wrinkled bi flattened or toward disorder, some mucosal surface of jaundice, gastric antrum visible to gray nodules some form polypoid or cauliflower-like bulge, more rigid texture, and most of the rat gastric erosions congestion or ulceration. Weining granule low, medium and high dose groups folds disorder, mucosal thinning or folds flat, hyperplastic nodules, mucosal yellow dye compared with the model group compared to a significant improvement (P <0.05 or P <0.01), as shown in Table 1. Table 1 rat gastric mucosa pathologic changes

2.3 gastric mucosal histological changes

    Model rats in 10 cases, five cases of moderate and severe ATP, 4 cases of the IM; Weining granule number of patients in the high dose group IM significantly reduced (P <0.05) compared with the model group compared; Weining granule The number of cases occurred in the high dose group of ATP, significantly lower (P <0.01), compared with the model group are shown in Table 2.

    2.4 rats telomerase activity

    Six cases of telomerase is activated in modeling drug effects, the model rats, gastric mucosal telomerase activity was inhibited Weining granule treatment, the Weining particles low dose group telomerase positive cases with the model group phase significantly lower (P <0.05), the Weining particles in the high dose group telomerase positive cases compared with the model group was significantly lower (P <0.01), as shown in Table 3. Table 2 groups rat gastric mucosa pathological changes Table 3 rat gastric mucosa telomerase activity

    2.5 rats in each group cell proliferation index (PI) apoptosis index (AI) changes

    Model mucosal cell proliferation enhanced apoptosis weakened; retinoic acid group, Weining particles, in the high-dose group, cell proliferation index (PI) was significantly reduced (P <0.01) compared with the model group, the apoptosis index (AI) was significantly higher (P <0.05), as shown in Figure 1.

    3 Discussion

    China is a high incidence of gastric cancer, the urban population of gastric cancer prevalence has declined, but a substantial increase in the number of rural population suffering from stomach cancer. At the same time, the proportion of young people suffering from stomach cancer in elevated. Medicine small side effect, compared with chemical drugs have certain advantages, new avenues for the development of non-toxic or low toxic anticancer drugs, a positive role to play in the prevention and treatment of gastric cancer [3,4]. Chinese medicine believes that gastric junction due by the lack of righteousness, Ecchymosis [5]. Weining granule Astragalus, Poria, Curcuma, wolfberry with Spleen Qi stasis role, for the treatment of gastric cancer and gastric cancer metastasis prevention. The modern pharmacological astragalus, Curcuma drugs can enhance the anti-tumor effect of dendritic cells to promote tumor-bearing host immune response, has a significant inhibitory effect [6,7]. Our previous study also found, the Weining particles can inhibit proliferation induced apoptosis in gastric cancer SGC  7910 cells, inhibits human gastric cancer SGC  7910 cells adhesion protein (FN), laminin inhibit cell motility and invasiveness, reducing people gastric cancer SGC  7910 cell matrix by metalloproteinase-2 (MMP  2) and matrix metalloproteinase  9 (MMP  9) the content of [8,9].

    The causes and mechanisms of modern medicine on the incidence of precancerous lesions is not yet fully understood, and may involve a variety of factors, such as Helicobacter pylori (HP) infection, tobacco, alcohol, irregular diet, salty or pickled foods (including high salt, Asia nitrate, etc.), with hot diet, the NSAID class of drugs to stimulate the duodenal juice reflux, immune, genetic, age and other factors, these causes persist or repeated gastric repeated injury and repair, the condition has become chronic first a variety of genetic abnormalities, was followed by abnormal cell metabolism, organized morphological abnormalities and even tumors [10, 11]. Recent studies found → precancerous cells → evolution of the cancer cells from normal gastric mucosa cells is a multi-factor, multi-gene action and complex process of multi-stage carcinogenesis [12, 13]. This process in cell metabolism, genetic instability (such as microsatellite instability, telomerase activation) abnormal changes in apoptosis and proliferation imbalance can occur malignant transformation. However, in the stage of precancerous lesions, although there are varying degrees of these changes, but these are reversible. Therefore, intervention in the treatment of precancerous lesions, prevent cancerous or put it into reverse to normal, is important to reduce the incidence of gastric cancer.

    Establishment of precancerous lesion model approach has the following five: (1) the gastric mucosa repeated damage modeling methods; (2) immune modeling method; (3) low-dose X-ray local modeling method; (4) active immunization combined with gastric mucosal damage modeling; (5) combined with the integrated modeling method carcinogens. The first four methods are complex or high cost or long cycle, it is difficult to promote the use of. To combine the the carcinogens comprehensive modeling method most commonly used by MNNG combination of precancerous lesions of the Joint Strike of other relevant factors, not only simulates human illness factors, but also shorten modeling time, after all, a better manufacturing gastric cancer precancerous lesion model. In this study, based on the successful establishment of the rat gastric precancerous lesions in experimental models, the naked eye Weining granule on gastric mucosal morphology change. Most of the model group hyperemia in rat gastric erosions or ulceration. Weining granule low, medium and high dose lesions have significantly improved, close to the normal group. The histopathological detection normal group rat gastric epithelial cells arranged in neat rows and integrity the gland morphology rules, the same size, the nucleus is located in the base of the glands, and occasionally inflammatory cell infiltration of the lamina propria, muscularis mucosa no hyperplasia to mucosal layer stretching. Model rats at 24 weeks (ie, the model is successful), the gastric mucosa was multifocal atrophic glands disordered arrangement, the base of the gland size difference is obvious, sizes, epithelial cell morphology and cell proliferation significantly deepening nuclear staining nuclear cytoplasm ratio increases, the phenomenon of a total wall, the muscularis mucosa extending to the mucous layer. Model rats in 10 cases, five cases of moderate and severe ATP, 4 cases of the IM; retinoic acid group 3 cases were mild or moderate of ATP, 6 patients with mild, moderate and severe CAG; Weining granule low, medium and high dose group IM ATP number of cases occurred significantly lower than the model group (P <0.05 or P <0.01), Weining granule can prevent the development of gastric carcinogenesis.

    Recent studies have found that telomeres and telomerase and cancer formation is closely related to [14]. When cells divide, the ends of chromosomes telomeres progressive shortening reaches a certain level, the cells showed the crisis, the telomerase is activated to maintain telomere length, cells have eternal life (immortalization). Telomerase is a ribonucleoprotein, a mainly composed of RNA and protein composition that it is different from the general DNA polymerase is a specificity of the reverse transcriptase. Telomerase can hexamer TTAGGG telomere repeat sequence. Due to the presence of telomerase, telomere length is maintained, the cells can be continued division and become immortalized cells that malignant cells. Most scholars believe that telomerase activation may be a key link in cell immortalization and malignant [15,16]. Telomerase activity in gastric carcinogenesis formation, play an important role in the development of superficial gastritis → atrophic gastritis → intestinal metaplasia → dysplasia → gastric cancer process in normal gastric mucosa → telomerase-positive expression rate was increasing trend [17, 18]. Thus, telomerase target clinics gastric carcinogenesis has important significance. Which in this experiment, the model in the modeling of drug action, six cases of telomerase is active, Weining particles after treatment the gastric mucosa telomerase activity was inhibited Weining granule by inhibiting end enzyme activity and thus the treatment of precancerous lesions .