Associations of Interleukin-1β with H. pylori-Induced Gastric Atrophy and Syndrome of Dampness-Heat in the Spleen and Stomach in Subjects with H. pylori-Related Gastric Diseases

H. pylori-related gastric diseases (HPGD) are a series of gastric mucosal benign and malignant lesions associated with H. pylori infection. Exploring the pathogenesis of HPGD will be of great significance to prevent and treat gastric malignancy. Traditional Chinese medicine (TCM) syndrome is the essence of TCM, reflecting the state of whole body. Potential similarities of TCM syndrome may provide a new perspective in understanding development and treatment of diseases. To seek an early warning signal for gastric malignant pathology and similarities of TCM syndrome from the viewpoint of molecular biology, we examined the relationships among H. pylori, gastric pathology, and TCM syndrome and effects of Interleukin-1β (IL-1β) gene polymorphisms and expression on gastric pathology and TCM syndrome in HPGD. The results indicated that detection of H. pylori with differentiation of TCM syndrome may have a predictive function to gastric pathology. H. pylori may lead to gastric atrophy via enhancing IL-1β mRNA expression, and IL-1β mRNA overexpression in gastric mucosa may be one of the generality characteristics for H. pylori-negative subjects with syndrome of dampness-heat in the spleen and stomach.


Introduction
Cancer is a common public health problem in the world. World Health Organization (WHO) reported that more than 9.6 million people have died of cancer in 2018, and gastric cancer (GC) was in the third place of the death rate of cancer. e development of GC is a complex, multistep, and multifactorial process. In 1992, based on evidence from pathology and epidemiology studies, Correa [1] put forward a human model of gastric carcinogenesis, which was chronic gastritis; atrophy; intestinal metaplasia (IM); and dysplasia. Infection with Helicobacter pylori (H. pylori) played a crucial role in the initial stages of gastritis and atrophy. H. pylori are a type of Gram-negative and microaerophilic bacteria which can attach to the surface of gastric mucosa and stimulate epithelial cells directly or indirectly to induce inflammation. us, H. pylori are believed to be the main cause of chronic gastric diseases. ese diseases are known as H. pylori-related gastric diseases (HPGD), which contain benign and malignant gastric disorders. However, not all subjects infected with H. pylori suffer from GC, implicating that there are other key factors in the process of GC.
Recently, genetic factors involved in DNA methylation and gene mutation are believed to contribute to GC development. Single nucleotide polymorphism (SNP) is one kind of mutations which might lead to abnormal gene expression and determine an individual's susceptibility to diseases. Inflammatory pathways run through all stages of HPGD. Interleukin 1β (IL-1β) is a proinflammatory mediator, which activates various immune and inflammatory responses in gastric mucosa after H. pylori infection [2]. IL-1B-511 (rs 16944) has been associated with various inflammatory and infectious disorders, including H. pylori infection [3], chronic atrophic gastritis [4][5][6], gastric ulcer [7], and gastric carcinoma [8]. However, to the authors' knowledge, there are scarce data regarding the relation of IL-1B-511 polymorphism and gastric IM and dysplasia. IL-1B-511 TT genotype has been reported to upregulate expression of IL-1β in gastric mucosa in healthy subjects [9]. erefore, it is necessary to explore systematically the relationship between IL-1B-511/IL-1β mRNA and pathological changes of gastric mucosa from normal to carcinoma.
A one-sided conclusion is easily obtained when disease development is analysed only from a single factor (e.g., pathogenic, genetic, or environmental factors). Based on holism, traditional Chinese medicine (TCM) puts forward the concept of "syndrome" defined as the reaction to or interaction with environmental and pathogenic factors for the host. Clinical physicians differentiate patients' syndrome through comprehensive analysis of signs and symptoms, determining the cause, nature, and location of the illness and the patients' physical condition, as well as treatment [10]. To some extent, TCM syndrome seems to have higher meaning than disease for the body. It has been reported that there is a close relationship between TCM syndrome and pathological changes in gastric diseases [11][12][13]. Gastric histopathology is regarded as the essence of gastric diseases in micro, while TCM syndrome as the whole in macro. However, there are few researches on association of TCM syndrome and gene polymorphism.
In this study, we analysed the relationship between gastric histopathology and TCM syndrome, as well as the association of gastric histopathology/TCM syndrome with some host factors (age, gender, and IL-1B-511 genotypes) and H. pylori, aiming to provide an early warning signal for gastric malignant pathology and to seek similarities of TCM syndrome. In addition, IL-1β mRNA levels in gastric mucosa were examined to investigate the molecular mechanism of gastric pathological changes and TCM syndrome.

Study Population.
All of the subjects were recruited consecutively from the Endoscopy centre of First Affiliated Hospital of Guangzhou University of Chinese medicine (Guangdong, China) from September 2011 to December 2016. ey comprised subjects undergoing a routine health check-up that included gastroscopy and patients with gastric disabling symptoms diagnosed as gastric inflammatory, gastric ulcer, or GC by gastroscopy. General information and clinical manifestations of subjects were collected. Several biopsy specimens were obtained from the antrum or the lesion location in the stomach. ese specimens were used for H. pylori detection, gastric mucosal histopathology, IL-1β gene polymorphisms, and mRNA detection. All samples were delinked and unidentified from their donors. Written informed consent was obtained from each individual before the study. All procedures followed in the study were approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Syndrome. TCM syndrome of  subjects was differentiated by two expert TCM doctors  according to guiding principles of clinical research on  Chinese traditional medicine and new medicine (trial edition) in 2002 [14], syndrome of dampness-heat in the spleen and stomach diagnose standards [15], spleen-qi deficiency syndrome diagnose standards [16], and WHO international standard terminologies on traditional medicine in the Western Pacific region [10]. Syndrome was identified as follows: (1) Syndrome of dampness-heat in the spleen and stomach (DHSS): main symptoms include yellow dense and slimy fur and sticky stool; secondary symptoms include stomach fullness, nausea, bitter and sticky taste, heaviness sensation of the body, thirst with a little intake of fluid, and poor appetite. (2) Liver-stomach disharmony syndrome (LSD): main symptoms include epigastric and hypochondriac dispending pain, stuffiness sensation in the chest with frequent sighing, and symptoms linked to mood; secondary symptoms include belching, gastric upset, acid vomiting, irritability, white slippery or thin yellow fur, and string-like pulse. (3) Spleen-qi deficiency syndrome (SQD): main symptoms include anorexia with indigestion, fatigue, postprandial abdominal distension, and pale tongue with fat or teeth-marked tongue; secondary symptoms include lassitude, bland taste in mouth with no thirst, sallow complexion, loose stool, and weak and thin pulse. (4) Syndrome of blood stasis in the stomach collateral (BSSC): main symptoms include epigastric stabbing pain that is aggravated by pressure in the epigastric region, dark red tongue, and purple spots on the tongue; secondary symptoms include greyish complexion and string-like and rough pulse. TCM syndrome is confirmed for each subject in the condition of two main symptoms or one main symptom with two secondary symptoms. If two or more than two kinds of syndromes occur in one subject, the main syndrome will be judged by subsequent discussion. In addition, we set nonsyndrome (NON): the subjects have no uncomfortable symptoms or abnormal signs, with pale red tongue, thin white fur, and no obvious abnormal pulse.

H. pylori Detection and Assessment of Gastric
Histopathology. Different methods for detection of H. pylori infection have different sensitivities and specificities [17,18]. To improve the accuracy for the diagnosis of H. pylori infection, rapid urease test (Guangzhou Beisiqi Reagent Co., LTD, Guangdong, China) and methylene blue staining (Guangzhou Chemical Reagent Factory, Guangdong, China) were performed in this study. e subject would be classified as H. pylori infection if at least one of the two methods was positive. Histopathological evaluation was performed by the hematoxylin and eosin method and examined by a single pathologist blinded to the participants' other information. According to the update Sydney system [19] and Morson's criteria of grading gastric dysplasia [20], degrees of gastric inflammation, atrophy, IM, and dysplasia were distinguished. e subjects with normal histology or just only mild inflammation were classified as the normal group (NOR).
ose with moderate-severe inflammation were divided into 2 Evidence-Based Complementary and Alternative Medicine the gastric inflammation group (GI). Gastric atrophy, IM, and dysplasia usually occur in one section, and subjects with severe dysplasia more easily suffer from GC than those with mild and moderate dysplasia. To further explore the differences among gastric pathologies, we determined that the subjects with only gastric atrophy were classified as the gastric atrophy group (GA); those with either IM or mild-moderate dysplasia were classified together as the gastric premalignant lesion group (GPL); and those with severe dysplasia were divided into the gastric severe dysplasia group (GSD). Patients with GC were classified as the gastric cancer group (GC). e PCR products were separated by electrophoresis on 1% agarose gel with Goldview fluorescent dyes. e PCR amplification was successful if only one single band of 241 bp was shown. Direct sequencing of PCR products was carried out by the dideoxy-chain termination method.

Quantitative Real-Time Polymerase Chain Reaction (Q-PCR) Assay.
Total RNA was extracted from the biopsy specimens using RNAiso Plus Kits (TaKaRa, AKA1202). cDNA was synthesized using a ReverTra Ace qPCR RT Kit (TOYOBO, FSQ-101). PCR amplification was performed with a three-step method using an ROX Reference Dye and SYBR Premix Ex TaqTM (TaKaRa, AK6006). Initial denaturation for cDNA was 95°C for 30 seconds. e amplification condition was 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 1 minute. e melt condition was 95°C for 15 seconds, 65°C for 1 minute, and 95°C for 15 seconds. e sequences of primers were the same as those of the IL-1B-511 genotyping primers as shown above. e level of IL-1β mRNA was normalized with β-actin as an internal control and compared with the data of one normal subject using the 2-ΔΔC(t) method.

Statistical
Methods. Stata 11.0 software was used for statistical analysis. All allelic distributions of IL-1B-511 were tested for deviations from their corresponding Hardy-Weinberg equilibrium. Age (per 10 years), gender, H. pylori, IL-1B-511 genotypes, and TCM syndrome distribution among gastric histopathological groups were summarized by proportions, and were examined using the chisquare test or Fisher exact probability test. Multinomial logistic regression estimated the adjusted (multivariable) relative risk ratio (RRs), 95% CIs, and P values. Because data did not obey normal distribution, levels of IL-1β mRNA between two groups were assessed by the Wilcoxon test. All values were two-sided, and statistical significance was defined as P < 0.05.

Characteristics of Subjects.
e study subjects consisted of 464 participants (264 males and 200 females). e mean age was 46.26 ± 12.95 years. All of them were divided into six histopathology groups through assessment of gastric histopathology ( Figure 1) and five TCM syndrome groups. H. pylori infection was examined by the rapid urease test and methylene blue staining ( Figure 2). 78.66% (365/464) subjects were positive for H. pylori infection. IL-1B-511 genotypes in gastric mucosa from 424 subjects were detected by the PCR-direct sequencing method (Figures 3 and 4). e frequencies of genotypes CC, CT, and TT of IL-1B-511 were 22.41% (95/424), 53.54% (227/424), and 24.06% (102/424), respectively. e alleles at the individual locus did not deviate from the Hardy-Weinberg equilibrium in this study, with nonsignificant χ 2 values (χ 2 � 2.14, P � 0.1435). In addition, levels of IL-1β mRNA in gastric mucosa were tested in 147 of 464 subjects (25 H. pylori-negative and 122 H. pylori-positive subjects).

Relationships between Gastric Histopathology and TCM
Syndrome. In all subjects, there were remarkable differences in frequencies of TCM syndrome between NOR group and other five histopathological groups (all P values were less than or equal to 0.001) and between the GI group and GPL group (P � 0.025). Further analyses showed that subjects with nonsyndrome in NOR group were the most among gastric histopathological groups, and the proportion of BSSC syndrome in the GPL group was higher than that in the GI group (10.88% vs. 3.75%) ( Figure 5(a)). To exclude the effects of H. pylori on gastric histopathology and TCM syndrome, we analyzed the distribution of TCM syndrome in different histopathological groups in H. pylori-negative and -positive subjects, respectively. In H. pylori-negative subjects, distribution of TCM syndrome in the NOR group was significantly different from that in the GI group (P < 0.001) and GPL group (P < 0.001), as well as that in the NOR group with H. pylori infection (P < 0.001) (Figures 5(b) and 5(c)). Proportion of H. pylori-negative subjects with nonsyndrome in the NOR group was much higher than that in the other histopathological group with or without H. pylori infection. Moreover, in H. pylori-positive subjects, significant difference of TCM syndrome distribution was observed between GI group and GC group (P � 0.025) ( Figure 5(c)). Specifically speaking, the rate of BSSC syndrome in the GC group was higher than that in the GI group (16.67% vs. 2.78%). Considering the effects of cofounding factors, we performed multinomial logistic regression. Table 3  To further evaluate roles of these variables on different histopathological stages, the latter histopathological group was compared with the former group, such as GI vs. NOR, GA vs. GI, GPL vs. GA, GSD vs. GPL, and GC vs. GSD by multinomial logistic regression. e results were shown in Table 4. Obviously, age, gender, and H. pylori had significantly important effects on gastric mucosa from normal to inflammatory (RRs were 1.47, 2.19, and 2.89). H. pylori infection was also a risk factor in gastric atrophy from inflammatory (RR � 5.51), while age played a crucial role in gastric severe dysplasia from gastric premalignant lesion (RR � 1.76). In the progress of gastric mucosa from atrophy to premalignant lesion, age might be a risk factor (RR � 1.29, P � 0.052).

IL-1B-511 Genotypes and Gastric
We used the NON group as a control to analyze relationships between covariables and TCM syndrome. As shown in Table 5

Relationships between Levels of IL-1β mRNA and Gastric
Histopathology/TCM Syndrome. In all subjects' detected levels of IL-1β mRNA in gastric mucosa, we demonstrated that IL-1β mRNA level in the GA group was dramatically higher than that in GI, GPL, GSD, and GC groups (All P values were less than 0.05) (Figure 6(a)). Also, in H. pyloripositive subjects, the median level of IL-1β mRNA in the GA group was the highest among histopathological groups and was significantly higher than that in the GPL group (82.14 vs. 4.63) (Figure 6(c)). However, no association between levels of IL-1β mRNA and gastric histopathology was observed in H. pylori-negative subjects (Figure 6(b)). ese results suggested that H. pylori-induced gastric atrophy was associated with IL-1β overexpression.   e asterisk ( * ) represents that P value is less than 0.05 and ( * * ) represents that P value is less than or equal to 0.001.
Evidence-Based Complementary and Alternative Medicine e relationship between IL-1β mRNA levels and TCM syndrome was also analyzed in our study. We found that in H. pylori-negative subjects, the median level of IL-1β mRNA in the DHSS group was significantly higher than that in the NON group (82.71 vs. 2.80; P � 0.0272), LSD group (82.71 vs. 3.37; P � 0.0142), and SQD group (82.71 vs. 1.06; P � 0.0281) (Figure 7(b)). However, no significant differences of IL-1β mRNA levels were observed among different TCM syndrome groups in H. pylori-positive subjects (Figure 7(c)) and all subjects (Figure 7(a)). ese results indicated that H. pylori-negative subjects with DHSS had a higher IL-1β expression.

Discussion
TCM, one of the oldest healing systems, plays an unreplaceable role in China and even the world. However, as the core of TCM, syndrome has been challenged by western medicine for a long time. It is essential to understand TCM syndrome from modern medicine and then to improve TCM. Nowadays, histopathology is the gold standard to diagnose various diseases and distinguish benign and malignant lesions. Both TCM syndrome and histopathology are the results of multiple factors acting on the host, reflect the current state of the host, and predict development of diseases.
us, there may be a relationship between TCM syndrome and histopathology.
TCM theory thinks that patients with long-term disease will easily form blood stasis. In this study, we found that in H. pylori-positive subjects, BSSC syndrome was significantly more common in the GC group than that in the GI group, which suggests that endoscopy should be performed carefully for BSSC patients with H. pylori infection to observe their gastric mucosa, and medicine with activating blood should be used appropriately for GC patients with H. pylori infection. Interestingly, only 5.56% of H. pylori-positive      [21] and the host's state. Both gastric pathology and TCM syndrome are results of multiple factors including host and environmental factors. In the present study, we focused on age, gender, H. pylori, IL-1B-511 genotypes, and IL-1β mRNA level which may participate in the formation of gastric pathology and TCM syndrome in subjects with H. pylori-related gastric diseases.
We showed that gastric histopathology and TCM syndrome were associated with age, gender, and H. pylori, but not IL-1B-511 genotypes. In detail, the risk of GI, GPL, GSD, and GC was increased with aging. Especially, age played an important role in the progress of gastric mucosa from normal to inflammatory and from GSD to GC. Although there was no statistical association, age had an increasing trend of gastric mucosa from GA to GPL. Moreover, H. pylori played a key role in the process of gastric mucosa from normal to inflammatory and from inflammatory to atrophy. ese results are the same as those of Correa's model. For TCM syndrome, compared with the NON group, age and H. pylori infection increased the risk of DHSS, LSD, SQD, and BSSC, which indicates that the state of human body will be changed from balance to imbalance with age increasing and H. pylori infection. When controlling age and H. pylori, we found that male gender raised the risk of GI, GSD, and GC and played an important role in the gastric inflammation from normal gastric mucosa. Also, male gender elevated the risk of DHSS, LSD, and BSSC. ese indicate gender may also be a factor in the formation of gastric pathology and TCM syndrome, which was probably related to sex hormones [22,23] and their receptors [24,25], as well as different lifestyle between males and females, such as smoking, frequent drinking, and bad dietary habits [26,27]. For IL-1B-511 polymorphisms, TT genotype with H. pylori infection has been reported to increase the risk of gastric atrophy in high prevalence region of gastric cancer (Shanxi) in China [5] and the risk of GC in population with Han nationality (Hubei) [28], and TT/CT genotypes could increase risk of GC in Chinese population (Jiangsu [29] and Guangdong [30]). However, no significant association between IL-1B-511 genotypes and gastric histopathology/TCM syndrome was found whether adjustment of age, gender, and H. pylori or not in our study where most of the subjects were from Guangdong. us, more evidence and clinical factors need * * * *  Evidence-Based Complementary and Alternative Medicine to be considered, such as haplotypes containing IL-1B-511 locus [7], lifestyle, and H. pylori strains [31]. We previously hypothesized that it was feasible to explore the formation mechanism of HPGD pathology and DHSS and other TCM syndromes from the inflammatory responses pathway and gastric microecology [32,33] and found that interleukin-12 (IL-12), interferon-c (IFN-c), granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulation upon activation normal T-cell expressed and secreted (RANTES) in gastric mucosa were involved in HPGD with DHSS pathology [34,35]. In the present study, we examined levels of IL-1β mRNA in gastric mucosa. e results showed that IL-1β mRNA was overexpressed in H. pylori-induced gastric atrophy. IL-1β has been reported to induce parietal cells atrophy by suppressing Sonic Hedgehog (Shh) gene expression and the release of intracellular calcium [36]. us, we speculate that H. pylori may induce gastric atrophy by IL-1β overexpression. It has been reported that IL-1β protein level in serum of rats with DHSS syndrome was higher than that in serum of normal rats, and heat-clearing and dampnesseliminating prescriptions reduced expression of IL-1β and improved the symptoms and signs [37,38]. is study also demonstrated that level of IL-1β mRNA in gastric mucosa of H. pylori-negative subjects with DHSS was significantly higher than those with nonsyndrome, LSD, and SQD. DHSS is thought be the status of struggle between the healthy qi and pathogenic qi, while nonsyndrome indicates the body is in a relative balance state, LSD is defined as dysfunction of the liver and stomach, and SQD is a pathological change characterized with impaired transporting and transforming function of the spleen. us, IL-1β overexpression might be a marker or a product of the host's struggle against pathogenic qi. Combined with our previous results, DHSS may be an external reflection of gastric mucosa inflammation.

Conclusions
In summary, we have shown that TCM syndrome occurs sooner than gastric pathological change after H. pylori infection. Under this condition, it is possible that TCM treatment helps prevent the development of gastric diseases. Our study also serves up an interesting perspective that combination of syndrome differentiation and H. pylori detection is likely helpful for predicting gastric histopathology to some extent. It remains unclear about the association of IL-1B-511 polymorphisms with gastric histopathology and TCM syndrome in patients with HPGD. But, we tentatively draw the conclusion that H. pylori may lead to gastric atrophy via enhancing IL-1β mRNA expression, and IL-1β mRNA overexpression in gastric mucosa may be one of the generality characteristics for H. pylorinegative subjects with DHSS.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.