Therapeutic mechanisms of integrated traditional Chinese and conventional medicine underlying its treatment of precancerous lesions of gastric cancer

Abstract

China has a high incidence of gastric cancer. Secondary prevention of gastric cancer is a major public health problem that must be solved urgently. Modern medicine focuses on the pathogenesis of precancerous lesions of gastric cancer (PLGC), and it has been found that there are a variety of abnormal gene expression patterns in PLGC. This study summarizes recent advances in our understanding of the therapeutic mechanisms of PLGC from various clinical studies; this will provide a reference for studying the therapeutic mechanisms of Traditional Chinese Medicine in the treatment of PLGC from the combined points of view of Traditional Chinese and Conventional medicine.

1. INTRODUCTION

According to global cancer epidemiological data from 2020, gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide, with almost half of the deaths occurring in China.¹ With an estimated 679 000 new cases and 498 000 deaths in 2015 in China,² GC ranked second among all malignant tumors for both incidence and mortality. GC is the result of a decades-long multi-step and multi-factorial process, which presents opportunities for interventions to prevent the disease. The process termed the “Correa model”³ starts as an inflammatory process in the antrum, usually associated with Helicobacter pylori (H. pylori) infection, which may progress toward a multi-focal chronic atrophic gastritis (CAG), followed by intestinal metaplasia (IM), dysplasia, and finally invasive carcinoma. Precancerous lesions of gastric cancer (PLGC) is a concept of histopathology that is often seen in patients with CAG and represents a pathological state that includes IM and dysplasia. Data from China showed that the proportion of lesions such as CAG and dysplasia that were confirmed by gastroscopy and pathological biopsy was as high as 30%.⁴ Some studies have found that the rate of mild dysplasia developing into cancer is 8.18%, while that rate for severe dysplasia is 72.09%; the rate for moderate dysplasia is between the two.⁵

Consequently, active interventions to block the development of PLGC into cancer is an important part of the secondary prevention of GC. Scientific research into the pathogenesis, clinical features, treatment measures, and therapeutic mechanisms of PLGC have been carried out for many years from the perspectives of both traditional Chinese and conventional medicine. With advances in science and technology, the understanding of diseases by modern medicine has been extended to the microscopic level. Conventional medicine is mainly used for treatments such as for anti-H. pylori infection, anti-bile reflux, anti-oxidation, inhibition of COX-2, and other comprehensive therapies, but effective treatments for PLGC are not currently available. Traditional Chinese medicine has achieved great clinical effect for PLGC. Jiang et al ⁶ analyzed 23 randomized controlled trials on treatments for chronic atrophic gastritis with integrated traditional Chinese and conventional medicine; the curative effect analysis of 2537 patients with PLGC showed that integrated traditional Chinese and conventional medicine could improve overall symptoms and pathological features such as glandular atrophy, IM, and atypical hyperplasia, and could delay or reverse the progression of atrophic gastritis. The combination of traditional Chinese and conventional medicine can organically unify the advantages of modern science and traditional medicine.⁷ Modern science can help to understand and clarify the esoteric and complex theories of Traditional Chinese Medicine, while the unique understanding of human life phenomena and the unique disease treatments of traditional medicine can enrich modern medical practices. Taking PLGC treatment as an example, the eradication of H. pylori is one benefit of conventional medicine; however, Traditional Chinese Medicine can also play a huge role by regulating genes to inhibit cell proliferation, promote apoptosis, correct abnormal methylation, and regulate cytokines and immune function. This review will synthesize the literature on the clinical treatment of PLGC with Traditional Chinese and conventional medicine, and summarize current research progress into the therapeutic mechanisms of PLGC treatment.

2. REGULATION OF CELL PROLIFERATION AND THE EXPRESSION OF APOPTOSIS GENES

An imbalance between apoptosis and proliferation plays an important role in the development of PLGC. Nuclear factor kappa-B (NF-κB) is a dimer composed of p65 and p50 in different forms, which participates in infection responses, inflammatory responses, regulation of immune responses, apoptosis, tumorigenesis, and development.⁸ Jin et al ⁹ found that the rates of positive NF-κB/p65 expression in chronic superficial gastritis, intestinal metaplasia, atypical hyperplasia, and GC were 0.0%, 36.7%, 76.7%, and 65.7%, respectively. The results suggest that NF-κB/p65 is activated in precancerous lesions, where it enters the nucleus to activate the transcription of downstream anti-apoptotic genes. B-cell lymphoma-2 (Bcl-2) is a known inhibitor of apoptosis. NF-κB can bind to a specific site on the Bcl-2 promoter and induce its expression during endoplasmic reticulum overload. Bcl-2 can inhibit apoptosis, while Bcl-2-associated X (Bax), which belongs to the same Bcl-2 protein family, binds to Bcl-2 to promote apoptosis; thus, Bcl-2 and Bax are intricately linked in apoptosis regulation. Cui et al ¹⁰detected the expression of NF-κB, Bcl-2, Bax, and the apoptosis index (AI) in 84 cases of GC and 54 cases of gastric atypical hyperplasia. In mild atypical hyperplasia and severe atypical hyperplasia, the expression rate of NF-κB and Bcl-2 protein gradually increased, while the expression rate of Bax protein gradually decreased. From mild to severe atypical hyperplasia, the AI gradually decreased, suggesting that NF-κB can regulate apoptosis by upregulating Bcl-2 expression and changing the Bcl-2 to Bax ratio. Through clinical trials, Xiao et al ¹¹ found that the Jianpi Hewei formula can effectively promote apoptosis and prevent GC to some extent by downregulating the NF-κB/Bcl-2 pathway. Pang et al ¹² found that Jianpi Xiaodu drink could promote apoptosis by downregulating Bcl-2 expression and upregulating Bax expression in the gastric mucosa of PLGC patients, which may be one of the mechanisms of its intervention.

Ki-67 is a nuclear antigen associated with proliferating cells; additionally, its function is closely related to the cell cycle phase of mitosis. Ki-67 is one of the important indicators used to detect the proliferative activity of tumor cells.¹³ Studies have shown that Ki-67 is karyotyped in chronic gastritis, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and GC, where the Ki-67 index is 17.39%, 37.50%, 56.25%, and 77.78%, respectively. These data indicate that Ki-67 may be useful for improving the detection rate of PLGC and early GC and for judging the severity and progression of GC.^14,15 Liu et al ¹⁶ enrolled 156 patients with PLGC in a randomized controlled trial. Patients in the control group were treated with routine conventional medicine and patients in the treatment group were treated with Traditional Chinese Medicine based on symptom pattern identification. After treatment, it was found that syndrome differentiation and treatment with Traditional Chinese Medicine could effectively reduce the abnormal Bcl-2 and Ki-67 expression. Using the random number table method, Wang et al ¹⁷ divided 117 patients with PLGC who met the inclusion criteria into Traditional Chinese and conventional medicine treatment groups. The 59 patients in the treatment group were treated with a Weizhuan'an prescription and the 58 patients in the control group were treated with a Weifuchun tablet. It was found that Ki-67 expression in the treatment group was lower than that in the control group after 6 months of treatment. This suggested that the Weizhuan'an formula could significantly reduce Ki-67 expression in the gastric mucosa.

3. REGULATION OF PROTO-ONCOGENES AND TUMOR SUPPRESSOR GENES

Abnormal activation of proto-oncogenes and loss or mutation of tumor suppressor genes can lead to abnormal cell proliferation and promote the progression of PLGC to GC. The proto-oncogenes closely related with PLGC are β-catenin, CyclinD1, C-erbB2, and C-met.¹⁸ Additionally, studies have shown that overexpression of β-catenin and CyclinD1, and the abnormal activation or amplification of C-erbB2 and C-met are closely related to PLGC occurrence and progression.^19,20 Many tumor suppressor genes have been studied in PLGC, including P53, P16, Runt-related Transcription factor 3 (Runx3), and phosphatase and tensin homolog deleted on chromosome ten (PTEN). The P53 gene is often mutated in the gastric mucosa; thus, the mutant p53 protein is usually detected by immunohistochemistry. Studies have shown that P53 protein expression is up-regulated in CAG, IM, dysplasia, and GC. Additionally, P53 overexpression is an early event in GC occurrence.²¹ The P16 gene can antagonize the effect of CyclinD1, and its hypermethylation plays an important role in the transformation from dysplasia to GC.²² Runx3 can inhibit cell differentiation and proliferation and can combine with β-catenin to regulate the transcription Wnt pathway target genes; Runx3 has been shown to be downregulated during gastric mucosal carcinogenesis.²³ PTEN has dual-specific protein phosphatase activity and can negatively regulate multiple signal transduction pathways including the AKT and ERK pathways through dephosphorylation. Additionally, PTEN can play a role in inhibiting growth, cell cycle regulation, promoting apoptosis, and inhibiting angiogenesis.²⁴

Gao et al ²⁵ reported that the rates of positive PTEN expression in dysplasia and GC were 71.4% and 46.6%, respectively. Additionally, Wang et al ²⁶ detected PTEN expression in gastric mucosa tissues of three different Traditional Chinese Medicine syndrome patterns of PLGC (spleen-stomach weakness pattern, Yin deficiency with internal heat pattern, and static blood obstructing stomach collateral pattern). It was found that the rates of positive PTEN expression were 85.71% in spleen-stomach weakness syndrome, 41.30% in Yin deficiency internal heat syndrome, 23.08% in stomach collateral stasis syndrome, and 96.16% in the healthy control group. The differences in PTEN expression in the different Traditional Chinese Medicine syndrome types of PLGC were statistically significant. These data provide a basis for the objectification of Traditional Chinese Medicine syndrome patterns of PLGC. Liang et al ²⁷ observed that PLGC patients were treated with Xinwei granules for deficiency of both Qi and Yin pattern, blood stasis and toxins. Moreover, it was found that Bcl-2 and C-myc protein expression in the gastric mucosa of the two groups were significantly decreased, while that of P53 and P16 were significantly increased, and the pathological state was significantly improved. This may also be one of the mechanisms through which Xinwei granules can ameliorate PLGC.

4. REGULATION OF MUCIN EXPRESSION

The mucin (MUC) family is a group of highly O-glycosylated glycoproteins, including transmembrane and secretory types of glycoproteins. Additionally, secretory MUCs, including MUC2, MUC5AC, and MUC6, are the primary components of gastric mucus, which is secreted by epithelial cells and protects the gastric mucosal epithelium from gastric acid, proteases, and pathogenic bacteria.²⁸ Different types of mucins are differentially expressed in different gastric tissues. In normal gastric mucosa, MUC5AC is distributed in the surface fovea epithelium and MUC6 is located in the deep glands. However, MUC2 is almost not expressed; the expression of MUC2 is often seen in the glands of intestinal metaplasia. Studies have shown that there is a certain correlation between the expression level of MUC and the subtypes of intestinal metaplasia of gastric mucosa.²⁹ In GC tissues, MUC5AC and MUC6 are weakly expressed and MUC2 is highly expressed. Previous studies have shown that the abnormal expression of MUCis associated with the occurrence and development of PLGC and GC.³⁰ Through clinical trials, Rao et al ³¹ found that the Jianpi Qingre Huayu formula increased MUC5AC protein expression in the gastric mucosa, improved symptoms of PLGC patients, enhanced protection from gastric mucosa, and reduced intestinal metaplasia and dysplasia. Through randomized controlled trials, Yan et al ³² concluded that the Yangyin Qingre Tongluo formula combined with conventional medicine was effective for treating PLGC patients and could significantly increase MUC5AC expression.

5. REGULATION OF INFLAMMATORY FACTORS

Rudolf Virchow first put forward the hypothesis that tumors originate from chronic inflammation.³³ This hypothesis emphasizes the close relationship between inflammation and the occurrence and development of tumors. Research into "inflammatory cancer trans-formation" has received great attention for the prevention and treatment of tumors and precancerous lesions. The interleukin (IL) protein family is produced by leukocytes and was initially regarded as a group of secretory proteins or signal molecules that regulate immune responses. Correlations between IL expression and cancer were later gradually explored.³⁴ The promotive cytokines IL-1β, IL-8, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) play important roles in PLGC. IL-1β can promote gastric epithelial proliferation and promote gastric mucosal atrophy by inhibiting the expression of gastric parietal cell-related factors.³⁵ In the gastric mucosal inflammatory response to H. pylori infection, IL-1β can reduce the clearance rate of cytotoxins and inflammatory products, lead to apoptosis, and promote the occurrence and progression of GC.³⁶ IL-8 was the first cytokine found to have a chemotactic effect on neutrophils. Currently, IL-8 is thought to be one of the key factors in the growth of GC, which has been confirmed by a large number of experimental studies.³⁷ IFN-γ has antiviral, antiproliferative, and immunomodulatory effects. It has been found that IFN-γ transgenic mice show gastric mucosal inflammation, metaplasia, and dysplasia.³⁸ TNF-α is a major inflammatory factor that not only mediates apoptosis and promotes cell proliferation, but also effectively inhibits gastric acid secretion, aggravates H. pylori infection, and promotes gastric mucosal atrophy.³⁹ Other studies have shown that TNF-α binds to target cell receptors through inflammatory mediators, activates a variety of signal pathways and related kinases to alter transcription, promotes inflammatory responses, and aggravates gastric mucosal injury.^40,41

In the study by Zhou et al ⁴² that included 140 PLGC patients, the treatment group was given Jianwei Xiaopi decoction and the control group was treated with Wenweishu capsules. After 4 months of treatment, IL-1β and TNF-α levels were lower in the treatment group than in the control group; conversely, IFN-γ levels were higher in the treatment group than in the control group (P < 0.01). This suggested that Jianwei Xiaopi decoction can regulate the inflammatory factors associated with PLGC, potentially blocking or reversing GC development. Additionally, it is beneficial for the prognosis of PLGC.

6. REGULATION OF EXOCRINE MICRORNAS (MIRNAS)

Recent studies have found that several novel genes and pathways play important roles in the occurrence and development of GC. MiRNAs show great potential for early screening and targeted gene therapy in GC.⁴³ MiRNAs are single-stranded non-coding RNAs, with lengths of 18 to 24 nucleotides. They participate in processes such as cell proliferation, differentiation, and apoptosis. Exosomes can mediate miRNA transport to make them stably expressed, and the expression of miRNAs is different in different tumors.⁴⁴ It has been found that miR-486p, miR-645, miR-624, miR-504, and hsa-miR-106b expression are upregulated, while that of miR-106b and miR-204 are downregulated in chronic gastritis and IM gastric mucosa-associated with H. pylori infection.⁴⁵ A study that detected plasma miRNAs in patients with PLGC and different stages of GC found that the concentrations of miR-16-5p and miR-19b-3p were slightly decreased in PLGC, and the degree of decrease in patients with early and advanced GC was positively correlated with the degree of GC invasion, which can be used as a potential biomarker for early screening and evaluating progression in GC.⁴⁶ Studies have shown that miR-133a and miR-421 play important roles in the occurrence, development, and prognosis of GC. They are highly expressed not only in the blood, but also in gastric juice.^47,48 Compared with blood, changes in the contents of gastric juice can better reflect the biological status of gastric tissue, which is helpful for understanding the degree of illness and determining patient prognosis.⁴⁹

Feng et al ⁵⁰ included 62 patients with atrophic gastritis and intestinal metaplasia. The control group was treated with verapamil tablets, and the treatment group was treated with Huatan Xiaoyu formula based on treatment in the control group. Three months after treatment, it was found that the miR-133a expression level in gastric juice (exocrine), the score of Traditional Chinese Medicine symptoms, the severity of gastric mucosal atrophy, and the scores of gastritis with IM and dysplasia in the observation group were significantly lower than those in the control group. The expression level of miR-421 in the gastric juice exocrine body was significantly higher than in the control group. These results suggest that Huatan Xiaoyu formula can alleviate the symptoms of patients with gastric precancerous lesions and reverse PLGC to some extent. Additionally, regulating miR-133a and miR-42 expression may be a relevant mechanism.

7. DISCUSSION

There seems to be no equivalent term for PLGC in Traditional Chinese Medicine. From the perspective of Traditional Chinese Medicine, based on the clinical symptoms of gastric pain, distension, and stuffiness in the hypochondrium, and anorexia, PLGC may be caused by Qi deficiency in the spleen and stomach, and blood stasis in the stomach collateral. Therefore, PLGC treatment primarily focuses on fortification of the spleen, replenishment of Qi, and resolution of stasis. Traditional Chinese and conventional medicine started from their respective theories, and in-depth theoretical discussions and scientific research have been carried out from many aspects, such as etiology, pathogenesis, evaluation of curative effect, and mechanistic research. There are many Traditional Chinese Medicine theories, from ancient times to the present, on the treatment of gastric precancerous lesions, and there are rich and detailed medical records to support the curative effects of these traditional treatments. Presently, the application of Traditional Chinese Medicine in PLGC treatment has been widely recognized as effective. However, this review found that the general level of evidence in Traditional Chinese Medicine-related clinical trials is not high, and there is still a lack of large-sample, multi-center, randomized controlled, double-blind clinical trials; mechanistic research and exploration are not deep enough and are mostly based on the rise and fall of indicators to speculate the mechanism. The authors believe that in the era of evidence-based medicine, obtaining high-quality research results with a high level of evidence is a major challenge in the study of the therapeutic mechanisms of Traditional Chinese Medicine in the treatment of gastric precancerous lesions. Generally, experimental animal research is ahead of clinical trials. However, we still must rely on large-sample, multicenter clinical trials to clarify the mechanisms of traditional medicine and to find clear therapeutic targets to provide strong theoretical support for clinical drug use and to guide further clinical trials. However, it is necessary to fully inherit and carry forward the quintessence of the classical theory of Traditional Chinese Medicine. This combined with modern advanced technology, and combining syndrome differentiation with disease differentiation, will improve symptoms and truncate reversals, optimize treatment plans, and maximize the unique advantages of Traditional Chinese Medicine.

8. ACKNOWLEDGMENTS

We thank James P. Mahaffey, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn/), for editing the English text of a draft of this manuscript.