Weipixiao ameliorates gastric precancerous lesions in a rat's model by regulating GSK3β and C-myc

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J Tradit Chin Med 2018 October 15; 38(5): 705-713 ISSN 0255-2922 . © 2018 JTCM. This is an open access article under the CC BY-NC-ND license.

RESEARCH ARTICLE TOPIC

Weipixiao ameliorates gastric precancerous lesions in a rat's model by regulating GSK3β and C-myc

Zeng Jinhao, Pan Huafeng, Guo Jing, Gong Daoyin, Cai Tiantian, Chen Xiaodong, Zhang Yi, You Fengming, Chen Longhui, Zhao Ziming, Liang Chao aa Zeng Jinhao, Guo Jing, Gong Daoyin, Zhang Yi, You Fengming, Liang Chao, Clinical Medical College, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China Pan Huafeng, Cai Tiantian, Chen Xiaodong, Chen Longhui, Guangzhou University of Chinese Medicine, Guangzhou 510405, China Zhao Ziming, Guangdong Provincial Institute of Chinese Medicine, Guangzhou 510095, China Supportted by the National Natural Science Foundation of China (No. 81804066; No. 81273739; No. 81473620) and the Research Project of Sichuan Provincial Administration of TCM (No. 2018QN022) Correspondence to: Prof. Pan Huafeng and Guo Jing, Guangzhou University of Chinese Medicine, Guangzhou 510405, China; Clinical Medical College, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China; [email protected]; [email protected] Telephone: +86- 20-36588339; +86-18980880167 Accepted: August 20, 2017

GSK3β, C-myc, Cylin E were evaluated by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. RESULTS: WPX could efficiently attenuate the pathological alterations of "non-progressive GPL" in rats. As expected, mRNA and protein levels of C-myc and Cylin E were up-regulated in model rats, while GSK3β expression down-regulated (P < 0.01). WPX treatment, especially at low dose, could significantly down-regulate the mRNA as well as protein levels of C-myc, and could lead to remarkable up-regulation of mRNA and protein levels of GSK3β in GPL rats (P < 0.05). However, no significant changes were observed in WPX-treated rats. CONCLUSION: Our findings suggested that WPX-mediated attenuation of GPL pathological alterations might be due to its regulatory effect on the expressions of GSK3β and C-myc, and on the dysregulation of Wnt/GSK3β pathway.

Abstract OBJECTIVE: To investigate the mechanism underlying the action of Weipixiao (WPX) in a rat's model with ameliorating gastric precancerous lesions (GPL).

© 2018 JTCM. This is an open access article under the CC BY-NC-ND license.

Keywords: Gastric precancerous lesions; GSK3β; C-myc; Cylin E; Weipixiao

METHODS: HPLC analysis was performed to identify the chemical constituents of WPX preparation. Sprague- Dawley rats were randomly assigned into control group, model group, vitacoenzyme group, high-dose WPX group (H-WPX), medium-dose WPX group (M-WPX) and low-dose WPX group (L-WPX). After modeling, the treated rats were administrated WPX or vitacoenzyme intragastrically for consecutive 10 weeks. Gene and protein expressions of JTCM | www. journaltcm. com

INTRODUCTION Gastric cancer (GC) is one of the major malignant tumors detrimental to human health, and it is a major cause of cancer-related mortality. An estimated 951 600 new gastric cancer cases and 723 100 deaths occurred worldwide in 2008, accounting for 6.8% of the total cases and 8.8% of total deaths.1 There is a high prevalence of gastric cancer in China, one of the major 705

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determinants for the high prevalence may be credited to a high incidence of gastric precancerous lesions (GPL).2,3 GPL are generally defined as gastric intestinal metaplasia (GIM) and gastric epithelium dysplasia (GED), and GPL may serve as the crucial transitional lesions from normal gastric mucosa to gastric cancer. Therefore, suppression or reversion of GPL may serve to lower the incidence of GC. It is well known that Wnt signaling pathway plays a key role in several developmental processes in embryonic state and also in the regulation of cell differentiation, proliferation, and apoptosis. Wnt signaling pathway comprises many genes that have been shown to be part of the signaling cascade, and their up-regulation or down-regulation could directly lead to the dysregulation of Wnt signaling pathway and thus lead to the occurrence and progression of numerous malignant tumors, including gastric cancer.5 GSK3β, C-myc and Cylin E are categorized as important components of the canonical Wnt/GSK3β pathway. The aberrant expression of these Wnt-related genes and thus the dysregulation of Wnt/ GSK3β pathway could initiate gastric carcinogenesis, not only by promoting cellular proliferation, but also by virtue of its opposing role in engendering programmed cell death. However, the differential expressions of these genes/ proteins in GPL are not fully understood. Weipixiao (WPX), a traditional Chinese herbal formula consisting of Huangqi (Astragalus Membranaceus Bunge), Taizishen (Pseudostellaria Heterophylla Pax), Baishu (Rhizoma Atractylodis Macrocephalae), Eshu (Curcuma phaeocaulis Val), Danshen (Salvia Miltiorrhiza Bunge) and Baihuasheshecao (Hedyotis Diffusa Willd), can fortify the spleen, dissipate blood stasis and exert detoxification. It exhibits favorable clinical efficiency in preventing and treating GPL.6 Our previous studies revealed that WPX could suppress and reverse GPL by attenuating cellular proliferation, increasing apoptosis, and by inhibiting inflammatory reactions. In present study, we used high performance liquid chromatography (HPLC) analysis to screen potential anti-GPL constituents of WPX separation. Then, we explored whether the Chinese herb preparation, WPX, had regulatory effect on mucosal expressions of Wnt-related genes/ proteins (GSK3β, C-myc and Cylin E) in GPL rats, aiming to elucidate the mechanism underlying the WPX's effects on GPL.

0111909). The experiments were conducted in Guangdong Provincial Institute of Traditional Chinese Medicine [specific-pathogen-free (SPF) laboratory animal facility: No. SYXK (Guangdong) 2010- 0059]. Drugs and reagents WPX contains the following herbs: Huangqi (Astragalus Membranaceus Bunge) 30 g, Taizishen (Pseudostellaria Heterophylla Pax) 15 g, Baishu (Rhizoma Atractylodis Macrocephalae) 15 g, Eshu (Curcuma phaeocaulis Val) 10 g, Danshen (Salvia Miltiorrhiza Bunge) 10 g, and Baihuasheshecao (Hedyotis Diffusa Willd) 30 g. The herbs were provided by the First Affiliated Hosipital of Guangzhou University of Chinese Medicine. The herbs were decocted in water twice, and then the filtrates were mixed and concentrated into a mixture of 1.5g/mL. All processes were performed by the Department of Pharmaceutical Preparation in Guangdong Provincial Institute of Traditional Chinese Medicine. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (No. ZG4T1-FP) was purchased from Tokyo Kabushiki Kaisha, Japan. C-myc rabbit mAb (No. ab32072) and Cylin E rabbit pAb (No. ab7959) were both supplied by Abcam, USA. GSK3β rabbit mAb (No. #5420S) was supplied by Cell Signaling Technology, USA. Platinum SYBR Green qPCR SuperMix-UDG was supplied by Life Technologies (Invitrogen), USA. High performance liquid chromatography (HPLC) analysis HPLC analysis was performed to identify the chemical constituents of WPX separation. Condition optimization of fingerprint: JADE-PAK ODS-AQ column (250 ´ 4.6 mm, 5 mm) and Inertsil ODS-SP column (4.6 ´ 150 mm, 5 mm) were used, with acetonitrile 0.1% phosphoric acid solution and acetonitrile 0.4% phosphoric acid solution as the mobile phase respectively, under full wavelength detection. The following chromatographic analysis conditions were determined: the separation was determined on Inertsil ODS-SP column (4.6 ´ 150 mm, 5 mm) with a mobile phase of acetonitrile (solvent A) 0.4% phosphoric acid solution (solvent B). For HPLC analysis, a 10 mL sample was injected into the column and eluted at a flow rate of 1.0 mL/min under room temperature. The detective wavelength was 203 nm. Grouping, modeling and treatment Using random number table method, animals were randomly divided into 6 experimental groups: control group (n = 10), model group (n = 15), vitacoenzyme group (n = 13, 0.2 g·kg-1·d-1), high-dose WPX group (H-WPX) (n = 13, 15g·kg-1·d-1), medium-dose WPX group (M-WPX) (n = 13, 7.5 g·kg-1·d-1) and low-dose WPX group (L-WPX) (n = 13, 3.75 g·kg-1·d-1). Based on the literature,7 the composite method was adopted to modeling the GPL rats. Briefly, All the rats, except for 10 control rats, were allowed to drink MNNG solu-

METHODS Ethical statement The study protocol and the handling of animals were in accordance with the international guidelines. Animals Seventy 7-week old male Sprague-Dawley (SD) rats, weighing 140-170 g, were provided by Experimental Animal Center, Sun Yat-sen University (certificate No. JTCM | www. journaltcm. com

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tion (200 μg/mL) ad libitum, and underwent hunger-satiety shift every other day, and during the satiety day the rats were free to ranitidine diet. At the end of 9th week, 2 random rats in the model group were sacrificed and examined for gastric precancerous lesions. At the beginning of 10th week, the treated rats were administered WPX or vitacoenzyme by gastrogavage for 10 consecutive weeks, while the control rats and the model rats were given distilled water by gastrogavage (1 mL/100 g body weight) daily.

Statistical analysis All data were presented as mean ± standard deviation ( xˉ ± s). Multiple group comparison was analyzed statistically by one-way analysis of variance (ANOVA), the comparison between two groups was performed with SNK method for the homogeneous variances, while the variances were heterogeneous, Dunnett's T3 method should be adopted. All analysis was performed using IBM SPSS 19.0 (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY, USA). A P value < 0.05 was considered significant.

Protein expression of GSK3β, C-myc and cyclin E by immunohistochemical method Paraffin-embedded gastric tissues were sliced into 3 μm sections. The sections were used for routine histopathological examination by hematoxylin and eosin (HE) staining and high-iron diamine-alcian blue-periodic acid Schiff (HID-AB-PAS) staining, and for protein expressions of GSK3β, C-myc and Cyclin E by EnVision immunohistochemical method. Five visual fields were randomly selected from each slice under light microscope (100 × ), and then images were acquired and quantitatively analyzed by Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD). Protein expression levels of GSK3β, C-myc and Cyclin E were all evaluated by scores of three indexes: mean of average optical density (AOD), sum of per area, object/total (PA), and mean of integrated optical density (IOD). Protein expression levels were considered to be up-regulation or down-regulation when any of the three indexes was increased or decreased.

RESULTS Chemical constituents of WPX Figure 1 shows the HPLC chromatograms of WPX test sample (A) and reference sample (B). The retention times of the major chemical constituents were 20.5 min (Calycosin-7- glucoside), 34.8 min (ginsenoside-Rg1), 48.3 min (ginsenoside-Rb1), 49.5 min (astragaloside Ⅳ), 59.0 min (atractylenolide Ⅲ), 71.7 min (atractylenolide Ⅱ), and 81.7 min (atractylenolide Ⅰ). Histopathological changes of the gastric mucosa by HID-AB-PAS staining In control rats, neutral mucins present in normal gastric epithelium were stained red (Figure 2A). In model rats, sialomucins present in small intestinal-type metaplasia (S-IM) were stained blue, and sulfomucins expressed in colonic-type metaplasia (C-IM) were stained brown (Figure 2B), suggesting that diffuse lesions of both S-IM and C-IM were found in model rats. In treated rats, the scope of IM lesion was reduced slightly in vitacoenzyme-treated rats (Figure 2C). Comparatively, the scope of IM lesion was reduced remarkably in all WPX-treated rats (Figure 2D-F).

Gene expressions of GSK3β, C-myc and cyclin E by qRT-PCR The mRNA levels of GSK3β, C-myc and Cyclin E were determined by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) method via IQ™5 real-time PCR detection system (Bio-Rad, USA). The primers were designed according to the sequences of GSK3β (GenBank accession no. NM_ 032080), C-myc (GenBank accession No. AY679730), Cyclin E (GenBank accession No. NM_001100821), and 18S (GenBank accession No. M11188) from Genbank database. All primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd., China (Table 1).

Histopathological changes of the gastric mucosa by H-E staining In control rats, normal structure/morphology of glands and epithelial cells was observed, and inflammatory infiltration in gastric epithelium was absent or minimal

Table 1 Primer Sequences of GSK3β, C-myc and Cyclin E Gene

PCR products length (bp)

Sequences of primers

Anneal temperature (℃)

F (5'-3'): ACGGCTACCACATCC 51 162 R (5'-3'): CAGACTTGCCCTCCA F (5'-3'): ACACCTGCCCTCTTC 51 147 GSK3β R (5'-3'): GTCTCCAGCATTAGTATCT F (5'-3'): ATCCTGTCCGTTCAAG 50 187 C-myc R (5'-3'): AAGTCCAAGTTCTGTCA F (5'-3'): GAACTGATGATGATGAAG 47 276 Cyclin E R (5'-3'): CCACTGATAACCTGAG Notes: PCR reaction conditions were as follows: 94 ℃ for 5 min; 45 cycles of 94 ℃ for 30 s, anneal at respective temperature for 30 s, and 72 ℃ for 50 s, followed by 72 ℃ for 7 min elongation. Melting curve analyses were performed on the amplified PCR products. The relative transcript amount of the target genes was normalized to that of 18S by using 2－△△Ct method. 18S

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A 17.5 15.0 12.5

40

50

60

7/81.731

30

6/71.709

20

70

80

2.5 0.0 min MPa

0 －10

17.5

12.5 7/81.401

10

5.0

15.0 6/71.805

20

5/59.140

30

7.5

B

3/48.342 4/50.110

40

10

2/35.385

0

3/48.266

5/59.041

10.0

60 mAU 50

MPa

1/20.696

350 325 300 275 250 225 200 175 150 125 100 75 50 25 0 －25

10.0 7.5 5.0 2.5

－20

0.0 0 10 20 30 40 50 60 70 80 min Figure 1 HPLC chromatogram of WPX test sample and reference sample A: test sample; B: reference sample. Peak: 1, Calycosin-7- glucoside; 2, ginsenoside-Rg1; 3, ginsenoside-Rb1; 4, astragaloside Ⅳ; 5-7, atractylenolide Ⅲ, Ⅱ, and Ⅰ, respectively.

(Figure 3A). In model rats, gastric epithelium was characterized by disorganized glandular architecture including marked crowding units and back-to-back glandular formation, and also manifested by cellular atypia having the following features: rounded, hyperchromatic and pleomorphic nuclei, increased nuclear-cytoplasmic ratio, loss of polarity, and increased karyokinesis. These pathological changes indicated that GED lesion was successfully induced (Figure 3B). In treated rats, GED lesion was improved in varying degrees, and the WPX treated rats, especially the L-WPX treated rats, showed a relatively remarkable improvement (Figure 3C-F).

Changes of C-myc mRNA expression In this study, level of C-myc mRNA expression in model group was significantly higher than that in control group (P < 0.01). Compared with model group, levels of C-myc mRNA expression in all WPX groups were significantly decreased (P < 0.01). Compared with vitacoenzyme, H-WPX and M-WPX groups, level of C-myc mRNA expression in L-WPX group was significantly decreased (P < 0.01 or P < 0.05). The data suggest that WPX could inhibit C-myc mRNA expression in GPL rats, and the L-WPX showed the highest inhibitory effect (Figure 5).

Changes of GSK3β mRNA Expression In this study, level of GSK3β mRNA expression in model group was significantly lower than that in control group (P < 0.01). Compared with model group, levels of GSK3β mRNA expression in all WPX groups were markedly increased (P < 0.01 or P < 0.05). Levels of GSK3β mRNA expression in both H-WPX and L-WPX groups were significantly increased when compared with vitacoenzyme group (P < 0.01). Level of GSK3β mRNA expression in H-WPX group was found significantly increased as compared to that in M-WPX group (P < 0.05). These results showed that WPX, especially at low dose, could markedly up-regulate GSK3β mRNA expression in GPL rats (Figure 4).

Changes of Cyclin E mRNA expression In this study, level of Cyclin E mRNA expression in model group was significantly higher than that in control group (P < 0.05), whereas no significant differences were seen among model, vitacoenzyme, and all WPX groups (P > 0.05). The result suggest that WPX might have no inhibitory effect on Cyclin E mRNA over-expression in GPL rats (Figure 6).

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Immunohistochemical analysis of GSK3β protein expression The protective effect of GSK3β on Wnt-associated gastric carcinogenesis and progression was well established. In the present study, scores of GSK3β protein expression (AOD, PA and IOD) were significantly de708

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B

A

B

C

D

C

D

E

F

E

F

Figure 2 Histopathological changes of the gastric epithelium in various groups (HID-AB-PAS staining, ×100) A: control group; B: model group; C: vitacoenzyme group; D: H-WPX group; E: M-WPX group; F: L-WPX group. Control group and model group: given with distilled water (1 mL/ 100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1·d-1); M-WPX group: treated with medium dose of WPX (7.5 g·kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao; HID-AB-PAS: high-iron diamine-alcian blueperiodic acid Schiff.

GSK-3β mRNA expression (fold)

Figure 3 Histopathological changes of the gastric epithelium in various groups (HE staining, × 400) A: control group; B: model group; C: vitacoenzyme group; D: H-WPX group; E: M-WPX group; F: L-WPX group. Control group and model group: given with distilled water (1 mL/ 100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1·d-1); M-WPX group: treated with medium dose of WPX (7.5 g·kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao; HE: hematoxylin and eosin.

creased as compared to that of control group (P < 0.01). Compared with model, vitacoenzyme and H-WPX groups, scores of GSK3β protein expression (PA or IOD) in L-WPX group were significantly decreased (P < 0.01 or P < 0.05). Therefore, it shows that L-WPX could markedly promote GSK3β protein expression in GPL rats (Table 2 and Figure 7).

1.0 0.8

bc

d

bce

a

0.6 0.4 0.2 0.0

del X me PX PX Mo nzy WP -W -W e L o H M ac Vit Groups Figure 4 GSK3β mRNA expression in gastric epithelial cells in various groups (n = 6) Control group and model group: given with distilled water (1 mL/100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1 ·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1 ·d-1); M-WPX group: treated with medium dose of WPX (7.5 g· kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g· kg-1·d-1). WPX: Weipixiao. aP < 0.01, compared with control group; bP < 0.01, cP < 0.05, compared with model group; dP < 0.01, compared with vitacoenzyme group; eP < 0.05, compared with M-WPX group.

Immunohistochemical analysis of C-myc protein expression A contribution of C-myc in gastric carcinogenesis has been implied from previous studies. In this study, scores of C-myc protein expression (AOD, PA and IOD) were significantly higher than that of control group (P < 0.01). Compared with model group, scores of C-myc protein expression in H-WPX (PA and IOD), M-WPX (IOD), and L-WPX (PA and IOD) were significantly decreased (P < 0.01 or P < 0.05). In addition, score of C-myc protein expression (PA) in L-WPX group was significantly decreased as compared to that of vitacoenzyme group (P < 0.05). The results revealed that WPX, especially at low dose, could markedly inhibit C-myc protein expression in GPL rats (Table 3 and Figure 8).

l

tro

n Co

and may trigger cancerization of epithelial cells. In this study, scores of Cyclin E protein expression (AOD, PA and IOD) in model group were significantly higher than that of control group (P < 0.01 or P < 0.05), whereas no significant differences in all indexes were shown among model, vitacoenzyme, and all WPX

Immunohistochemical analysis of cyclin E protein expression Aberrant expression of Cyclin E disrupts the cell cycle JTCM | www. journaltcm. com

1.2

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C-myc mRNA expression (fold)

Zeng JH et al. / Research Article 10 9 8 7 6 5 4 3 2 1 0

groups (P > 0.05). Thus, WPX might have no inhibitory effect on Cyclin E protein expression in GPL rats (Table 4 and Figure 9).

a

b

b

DISCUSSION bcde

Wnt signaling pathway has a key role in normal embryonic development and wound repair but is largely quiescent in normal physiologic state apart from in pathological state, such as carcinogenesis. Different intrinsic and extrinsic factors, such as chronic inflammation,8 and Helicobacter pylori (Hp) infection,9 may be closely correlated to the abnormal expression of Wnt-related genes and the dysregulation of Wnt signaling pathway. Thus, inappropriate activation of Wnt signaling pathway via dysfunction in core pathway components, such as GSK3β, C-myc and Cyclin E, is firmly established as the critical factor in the development of gastric cancer. GSK3β is evaluated as a central molecule necessary for the induction of β-catenin degradation and thus the inhibition of Wnt signaling pathway activation.10, 11 In addition, GSK3β is a key tumor- suppressive gene and is also crucial to the pathogenesis of gastric cancer. A study12 reported that inhibition of GSK3β could stimulate COX-2 over-expression in gastric cancer cells, which might be involved in the underlying mechanisms of gastric cancerous progression. Thus, the deficiency or down-regulation of GSK3β may contribute to the dysregulation of Wnt signaling in gastric cancer. Here, we demonstrated that mRNA and protein levels of GSK3β were significantly decreased in precancerous tissues when compared with those of normal tissues. C-myc is an important regulator of a wide array of cellular processes, including cell cycle, cell growth, differentiation, apoptosis and transformation, and even angiogenesis.13, 14 Furthermore, C-myc is also identified as a downstream target gene mediated by Wnt/GSK3β pathway. Recent studies 15,16 revealed that C-myc amplification/over-expression was frequently found in specimens from patients with gastric cancer and is positively correlated with advanced tumor stage, lymph node metastasis as well as poor survival time. The critical role of

ol ntr

e del PX PX PX -W Mo enzym -W -W M L H o ac Vit Groups Figure 5 C-myc mRNA expression in gastric epithelial cells in various groups (n = 6) Control group and model group: given with distilled water (1 mL/100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1 ·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1 ·d-1); M-WPX group: treated with medium dose of WPX (7.5 g· kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g· kg-1·d-1). WPX: Weipixiao. aP < 0.01, compared with control group; bP < 0.01, compared with model group; cP < 0.01, compared with vitacoenzyme group; dP < 0.05, compared with H-WPX group; eP < 0.01, compared with M-WPX group.

Cyclin mRNA expression (fold)

Co

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

a

l

del PX X me PX Mo WP M-W nzy e -W o L H c a Vit Groups Figure 6 Cyclin E mRNA expression in gastric epithelial cells in various groups (n = 6) Control group and model group: given with distilled water (1 mL/100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1·d-1); M-WPX group: treated with medium dose of WPX (7.5 g·kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao. aP < 0.05, compared with control group. tro

n Co

Table 2 Scores of GSK3β protein expression in gastric epithelium in various groups ( xˉ ± s) Group

n

AOD

Control

9

0.137±0.037

0.166±0.079

35.897±11.731

IOD

Model

9

0.074±0.040a

0.029±0.026a

7.218±5.367a

Vitacoenzyme

9

0.085±0.021

0.034±0.023

9.419±4.016

H-WPX

9

0.102±0.028

0.053±0.019

16.088±6.837

M-WPX

9

0.108±0.036

0.058±0.035

17.414±8.992

PA

0.113±0.037 0.110±0.040bcd 25.521±10.369be L-WPX 9 Notes: control group and model group: given with distilled water (1 mL/100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1·d-1); M-WPX group: treated with medium dose of WPX (7.5 g·kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao. aP < 0.01, compared with control group; bP < 0.01, compared with model group; cP < 0.01, eP < 0.05, compared with vitacoenzyme group; dP < 0.05, compared with H-WPX group. JTCM | www. journaltcm. com

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B

C

D

E

F

Cyclin E expression may be regulated by Wnt signaling pathway.19 Several studies revealed that Cyclin E overexpression was observed in most cases of gastric cancer, and it was significantly associated with intestinal Lauren classification, size of the tumor (larger than 5 cm), advanced stages, and lymphatic and vascular invasion.20, 21 In this study, dysfunction of C-myc and Cyclin E is not only one of the hallmarks in gastric cancer, but in gastric precancerous lesions as well, as proved by our RT-qPCR and immunohistochmistry analysis. In this study, up-regulated gene and protein expressions of C-myc and Cyclin E, and down-regulated gene and protein expressions of GSK3β were found in gastric mucosa from GPL rats. These results demonstrated that the abnormal expressions of the Wnt-related genes/proteins and thus the dysregulation of Wnt/ GSK3β pathwaymay be prominent in oncogenic transformation of gastric epithelium, strongly suggesting that the states of abnormality of cellular differentiation, increased cell proliferation and decreased cell apoptosis may be present in precancerous gastric mucosa. In addition, as the lesions progressed gradually from atrophic gastritis, to intestinal metaplasia, and to dysplasia (the so-called Correa pathway),22 an increased level of C-myc gene/protein expression and a decreased level of GSK3β gene/protein expression were observed in GPL rats. Thus, down-regulated expression of GSK3β may induce gastric tumorigenesis, in combination with up-regulated expression of C-myc. In accordance with the Traditional Chinese Medicine theory, GPL is conceptually referred to as "gastric stuffiness", of which the major manifestations were abdominal distension, stomach duct pain or gastric upset, stomach reflux or vomiting, hiccup, diarrhea or constipation, poor appetite, and weight loss. The principal pathogenesis of GPL may include the following three aspects:23 spleen-stomach weakness, blood stasis in the stomach collateral vessels, and pathogenic toxins dormant in the body. So the herbal preparation WPX is developed to fortify the spleen and invigorate the stomach, to dissipate the blood stasis from stomach collateral vessels, and to remove the pathogenic toxins from the body. In our previous clinical practice,6 WPX shows obvious effects at relieving the clinical symp-

Figure 7 Expression of GSK3β in gastric mucosal epithelial cells in various groups (immunohistochemical method, × 100) A: control group, GSK3β was mainly expressed in the cytoplasm, and GSK3β positive cells were distributed diffusely in gastric epithelium. B: model group, GSK3β was sparsely expressed in normal gastric epithelium. C: vitacoenzyme group; D: H-WPX group, treated with high dose of WPX (15 g·kg-1·d-1) E: M-WPX group, treated with medium dose of WPX (7.5 g·kg-1·d-1); F: L-WPX group, treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao. In the treated groups, there were more GSK3β positive cells than that in model group, of which the change was the most obvious in L-WPX group.

C-myc in gastric cancer is further supported by another study,17 which revealed that C-myc could promote the growth and proliferation of normal gastric cells, and that knockdown of C-myc could restrain the proliferation of normal gastric cells and induce apoptosis. Cyclin E is a pivotal regulator at the G1/S phase cell cycle transition in higher eukaryotes, premature expression of Cyclin E advances entry into S phase, whereas lack of Cyclin E prevents entry into S phase.18 Furthermore, silencing β-catenin gene may induce the changes of cell cycle and Cyclin E expression, suggesting that

Table 3 Scores of C-myc protein expression in gastric epithelium in various groups ( xˉ ± s) Group

n

AOD

Control

9

0.000±0.000

Model

9

0.117±0.040

Vitacoenzyme

9

H-WPX

IOD

PA

0.000±0.000

0.000±0.000 0.173±0.067

40.778±11.712a

0.104±0.042

0.138±0.049

32.535±13.859

9

0.080±0.022

0.085±0.022

19.633±8.368d

M-WPX

9

0.094±0.016

0.094±0.026

23.550±7.660b

L-WPX

9

0.077±0.012

0.072±0.017bc

17.675±5.656d

a

a

b

Notes: control group and model group: given with distilled water (1 mL/100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1·d-1); M-WPX group: treated with medium dose of WPX (7.5 g·kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao. aP < 0.01, compared with control group; bP < 0.05, dP < 0.01, compared with model group; cP < 0.05, compared with vitacoenzyme group. JTCM | www. journaltcm. com

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October 15, 2018 | Volume 38 | Issue 5 |

Zeng JH et al. / Research Article

A

B

C

D

E

F

markable improvement of S-IM lesion than that of C-IM lesion. We also found that WPX wasmarkedly beneficial to improving gastric epithelium with GED lesions-at least the lesions of indefinite for dysplasia (I-GED) and low-grade dysplasia (L-GED). However, lesion of high-grade dysplasia (H-GED) was observed in one case of M-WPX treated rats, which may be credited to the fact that H-GED is a "more aggressive GPL"-a high similarity was found between H-GED and early gastric cancer in terms of cell proliferative activity and cell atypia, so the pathological changes of some H-GED cases might be difficultly reversed. Therefore, WPX could at least efficiently attenuate the "non-progressive GPL" pathological alterations. In the present study, L-WPX was showed to be more effective in inhibiting the gene/protein expression level of C-myc, promoting the gene/protein expression level of GSK3β, in contrast with those of H-WPX and M-WPX. However, our previous studies revealed that H-WPX is more superior to M-WPX and L-WPX in blocking gastric precancerous lesions, suppressing cell proliferation and promoting apoptosis. We speculate that the inconsistency might be due to the different treatment duration (10 weeks in the present study, 4 weeks in the previous studies). The results suggest that WPX, at low dose, may be more ideal for long-term intervention of GPL when compared with that at high and medium doses. We will explore and validate the correlations among treatment dosage, intervention duration and therapeutic effect in WPX-treated GPL in further studies. However, WPX showed no regulatory effect on Cyclin E protein and gene expression, which might be attributed to the limitation of sample number, or to the fact that Cyclin E may not be the potential target of WPX. In this study, however, some insufficiencies still existed. For example, limitation of sample number, and intermolecular interactions among GSK3β, C-myc and Cyclin E were not investigated. These limitations should be considered in future studies. In conclusion, our findings suggested that WPX has regulatory effect on the expressions of GSK3β and C-myc, and on the dysregulation of Wnt/GSK3β pathway, thereby inhibiting cell hyper-proliferation in GPL

Figure 8 Expression of C-myc in gastric mucosal epithelial cells in various groups (immunohistochemical method, × 100) A: control group, GSK3β was mainly expressed in the cytoplasm, and GSK3β positive cells were distributed diffusely in gastric epithelium. B: model group, GSK3β was sparsely expressed in normal gastric epithelium. C: vitacoenzyme group; D: H-WPX group, treated with high dose of WPX (15g·kg-1·d-1) E: M-WPX group, treated with medium dose of WPX (7.5g·kg-1·d-1); F: L-WPX group, treated with low dose of WPX (3.75g·kg-1·d-1). WPX: Weipixiao. In the treated groups, the number of C-myc positive cells was fewer than that of model group, of which the change was the most obvious in L-WPX group.

toms, improving the gastric precancerous state (through gastroscopic and pathohistological examination), and partially eradicating Helicobacter pylori in patients with GPL. As expected, diffuse IM and GED lesions could be observed in model rats, indicating that the GPL rat model was successfully established. The present study demonstrated that WPX exerted favorable therapeutic effect on gastric epithelium with both small intestinal-type metaplasia (S-IM) and colonic-type metaplasia (C-IM). Additionally, WPX-treated rats showedre-

Table 4 Scores of Cyclin E protein expression in gastric epithelium in various groups ( xˉ ± s) Group

n

AOD

PA

IOD

Control

9

0.027±0.029

0.004±0.005

3.827±6.508

Model

9

0.140±0.035a

0.152±0.101b

33.008±14.826a

Vitacoenzyme

9

0.121±0.045

0.128±0.059

29.410±9.639

H-WPX

9

0.118±0.033

0.083±0.043

22.088±8.988

M-WPX

9

0.112±0.032

0.085±0.042

25.191±10.256

L-WPX

9

0.101±0.043

0.066±0.025

19.308±11.463

Notes: control group and model group: given with distilled water (1 mL/100 g body weight) daily; vitacoenzyme group: administered with vitacoenzyme (0.2 g·kg-1·d-1); H-WPX group: treated with high dose of WPX (15 g·kg-1·d-1); M-WPX group: treated with medium dose of WPX (7.5 g·kg-1·d-1); L-WPX group: treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao. aP < 0.01, bP < 0.05, compared with control group. JTCM | www. journaltcm. com

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Zeng JH et al. / Research Article

A

B 8

9 C

D 10

11 E

F 12

Figure 9 Expression of cyclin E in gastric mucosal epithelial cells in various groups (immunohistochemical method, × 100) A: control group, GSK3β was mainly expressed in the cytoplasm, and GSK3β positive cells were distributed diffusely in gastric epithelium. B: model group, GSK3β was sparsely expressed in normal gastric epithelium. C: vitacoenzyme group; D: H-WPX group, treated with high dose of WPX (15 g·kg-1·d-1) E: M-WPX group, treated with medium dose of WPX (7.5 g·kg-1·d-1); F: L-WPX group, treated with low dose of WPX (3.75 g·kg-1·d-1). WPX: Weipixiao. In the treated groups, the number of Cyclin E positive cells was relatively fewer than that of model group, of which the change among the groups showed no significant difference.

13

rats, which might be involved in the mechanism underpinning WPX's anti-GPL action.

17

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