Efficacy and Safety of Zuojin Pill for the Treatment of Chronic Nonatrophic Gastritis: A Randomized Active-Controlled Clinical Trial

Objective Zuojin pill (ZJP) is used as the classical prescription for a wide variety of digestive diseases. However, there is a lack of direct evidence for its use in the treatment of chronic nonatrophic gastritis (CNG). In particular, there is a lack of rigorous trials of randomized controlled designs. In this study, a randomized active-controlled clinical trial was performed to verify the efficacy and safety of ZJP in detail. Methods Patients with CNG were divided into the ZJP group and the Marzulene-S granule group. Patients were enrolled from September 2019 to February 2021 (ChiCTR2000040549). Endoscopy and histology scores were evaluated as the primary outcome measure. The Helicobacter pylori positive rate and the disappearance rate of symptoms were also measured to reflect the outcomes. Finally, adverse events were also calculated as the index of safety. Results A total of 68 eligible patients were enrolled in this trial and randomly divided into two groups with baseline comparability. ZJP was able to improve the red plaques as well as bile reflux scores compared with Marzulene-S granule (P=0.043 and P=0.019, respectively). Moreover, it also remarkably alleviated the active chronic inflammation score (P=0.043). However, there was no difference between the Helicobacter pylori positivity rate (P=0.752). The symptom scores of abdominal distension (P=0.004), belching (P=0.010), and loss of appetite (P=0.019) were alleviated by ZJP, but nausea and vomiting were not (P=0.616). ZJP can also be considered safe with no obvious adverse effects. Conclusion ZJP might decrease mucosal injury and alleviate symptoms in CNG. In addition, more large-scale clinical trials should be carried out to further confirm its clinical efficacy and safety.


Introduction
Chronic gastritis refers to the chronic infiltration of inflammation in the gastric mucosa and is the most common disease worldwide. It can be divided into three types: nonatrophic gastritis (CNG), atrophic gastritis (CG), and other special types. Recent data from the Digestive Endoscopy Branch of the Chinese Medical Association indicated that approximately 49.3% of patients with upper gastrointestinal symptoms were diagnosed with CNG. Further evidence suggested that CNG can develop into CG without proper treatment. Patients who had CG with premalignant gastric lesions were shown to carry a remarkable risk of gastric cancer within 10 years of follow-up. Even in the short term, symptomatic patients might have a decreased quality of life due to nonspecific dyspeptic symptoms, such as epigastric discomfort, belching, distention, nausea, and even loss of appetite. However, there is still a lack of a specific medicine for CNG treatment due to the complex pathogenesis. Helicobacter pylori (Hp) infection is believed to be the primary cause of CNG, and therapy involving the eradication of Hp is applied worldwide. Clarithromycin-containing regimens are recommended as first-line drugs. However, the increasing rate of antibiotic resistance has already influenced the treatment of CNG [1]. Moreover, proton pump inhibitors (PPIs), such as omeprazole, esomeprazole, and lansoprazole, are also important medicines that could reduce the damage to the mucosa that is induced by gastric acid. Studies have indicated that PPIs are associated with a series of adverse effects, including osteoporosis, hypomagnesemia, Clostridium difficile colitis, and cardiovascular morbidity [2,3]. In addition, bismuth quadruple therapy is also widely employed as a first-line treatment. However, current studies have suggested that it might cause short-term dysbiosis of the gut microbiota and lead to adverse effects [4]. Specific medicine with ideal efficacy that can improve the quality of life is urgently needed for CNG patients.
It has long been recognized that traditional medicine still demonstrates unique therapeutic characteristics in the modern clinical system [5,6]. Artemisinin, derived from Artemisia annua L. (Qinghao), is one of the most commonly used medicines to efficiently fight against malaria. e original plant was first recorded by Ge Hong in A Handbook of Prescriptions for Emergencies and was applied to relieve malaria symptoms. It is widely thought to be a gift from traditional Chinese medicine to the world [7]. Apart from artemisinin, Maxingshigan decoction and Yinqiao powder have also been recently used to fight against complex diseases. ese two prescriptions are thought to be antiplague formulae in traditional Chinese medicine. In 2011, the combination of these two formulae was demonstrated to reduce the fever resolution period of patients with H1N1 influenza virus infection compared with oseltamivir in a prospective randomized controlled trial [8]. Lianhuaqingwen capsule, one type of traditional Chinese patent medicine that is made from the combination of Maxingshigan decoction and Yinqiao powder, was further confirmed to be effective in alleviating coronavirus disease 2019 (COVID-19) pneumonia in the clinic [9]. erefore, it could serve as a crucial source for drug development from traditional Chinese medicine.
Zuojin pill (ZJP) was initially created by Danxi Zhu in his Danxi's Experiential erapy, which is a well-known formula that has been used since the 15th century. Two kinds of herbal medicine containing the root of Coptischinensis Franch. and the fruit of Euodia ruticarpa (A. Juss.) Benth. are involved in this prescription at a ratio of 6 : 1 (w/w). It is used for the pattern of liver fire invading the stomach, which represents specific symptoms, including heartburn, dyspepsia, vomiting, and abdominal distension, in traditional Chinese medicine. is pattern occurs in a wide variety of digestive diseases in modern clinics [10]. Hence, it is recognized as the principle medicine for the treatment of patients suffering from gastritis, gastroesophageal reflux disease, gastric ulcer, and even gastric carcinoma [11]. Several preclinical pharmacologic experiments also suggested that the Zuojin pill could significantly reduce gastric mucosal injury by inhibiting the regulation of the NF-lB signaling pathway and reducing the inflammatory response [12,13]. All these studies provide indirect data that confirm the therapeutic efficacy of the Zuojin pill on CNG. However, there is a lack of direct evidence, especially with rigorous trials of randomized controlled designs. In this study, an open-label, randomized controlled trial was performed to verify the efficacy and safety of the Zuojin pill in detail. e results will definitely guide clinical applications.

Endoscopic and Histopathological Diagnosis.
Endoscopic and histopathological examinations were the main references for CNG diagnosis. e essential characteristics of CNG are red plaques with punctuates, patches, and striae. Coarse and uneven mucosa, hemorrhagic spots or plaques, oedematous mucosa, and exudates without atrophic changes were also seen under endoscopy. Furthermore, no atrophic changes or intestinal metaplasia were found in the histopathological examination of the gastric mucosa.

Inclusion Criteria.
e inclusion criteria were as follows: participants were diagnosed with CNG according to the Chinese Consensus on Chronic Gastritis; participants were diagnosed with liver fire invading stomach syndrome, according to TCM syndrome differentiation; participants were between 20 and 70 years of age; participants voluntarily provided written informed consent.

Exclusion Criteria.
e exclusion criteria were as follows: patients who underwent gastric-related surgery; patients with atrophic changes, intestinal metaplasia, or suspected malignant changes in the gastric mucosa; patients who also had other systemic diseases in the heart, liver, lung, kidney, or blood system; female patients who are planning to have a baby, are currently pregnant, or are lactating; patients with life-threatening illnesses, such as tumors and AIDS; patients who received other kinds of medicine in the 2 weeks Evidence-Based Complementary and Alternative Medicine before recruitment; and patients who were judged as inappropriate to be enrolled in the trial by investigators.

Rejection Criteria.
e rejection criteria were as follows: patients who were misdiagnosed; patients who did not take the proper drug dosage; and patients who could not complete the follow-up.

Withdrawal Criteria.
e withdrawal criteria were as follows: patients who quit the clinical trial voluntarily and patients who were intolerant to the Zuojin pill or conventional therapy. Adverse events occurred during the trial. ese adverse events included severe hepatic injury, malignant tumors, or hemorrhage of the digestive tract, and thus, a patient with these events was unlikely to complete the procedure.

Intervention.
Participants assigned to the control group received Marzulene-S granule (2.01 g/day, three times a day, for 12 weeks). One granule of Marzulene-S weighed 0.67 g and contained 663.3 mg L-glutamine and 2.0 mg sodium gualenate. Participants in the treatment group received the Zuojin pill (6.0 g/day, twice a day, for 12 weeks). Zuojin pill was available from the Department of Pharmacy in the Fifth Medical Center of PLA General Hospital. A chemical analysis was performed to ensure the homogeneity of the prescription. During the treatment, a healthy lifestyle with a light diet, regular work and rest, and proper exercise were required, and smoking, alcohol consumption, and the consumption of spicy foods were prohibited. Patients with Helicobacter pylori also received clarithromycin-containing regimens for eradication. A follow-up was scheduled every 4 weeks following the treatment. Patients received conventional treatment if they did not obtain full recovery of CNG after the trial.

Primary Outcome.
e score of endoscopy evaluation, involving red plaques, erosion, hemorrhage, and bile reflux, was compared between the control group and the treatment group (Supplementary Table 1). e grade of histopathological changes, including chronic inflammation and active chronic inflammation, was also evaluated (Supplementary Table 2). e standard was set according to the Chinese Consensus on Chronic Gastritis.

Secondary Outcomes
(1) Helicobacter pylori positive rate. e Helicobacter pylori positive rate in patients was also evaluated for the treatment.
(2) Symptom scores. Changes in each symptom score, including abdominal distension, belching, nausea, vomiting, and loss of appetite, were compared to reflect the quality of life (Supplementary Table 3).

Subgroup Analysis. Subgroup analysis based on
Hp infection was also conducted with all the outcome measures.

Safety Outcome.
Patients were interviewed every 4 weeks to record adherence and identify adverse events. Routine tests, including blood, urine, and stool tests, electrocardiogram, and liver and renal function tests, were performed at baseline. If the adverse events were relevant to the medication, patients were followed until their adverse events were resolved or there was no clinical significance.

Preparation and HPLC-MS/MS Analysis of Zuojin Pill.
e preparation and HPLC-MS/MS analysis of the Zuojin pill were conducted according to a previous method.

Preparation of Zuojin Pill Extracts and Standard
Samples.
e root of Coptis chinensis Franch. and the fruit of Euodia ruticarpa (A. Juss.) Benth. ley were weighed and added at 6 : 1 ratio. ey were soaked for 30 min in pure water (1/10, w/v). After that, it was extracted twice by heating (1 h at a time). e filtrate was rotationally evaporated, condensed, and dried into a dry powder for preservation. e weight ratio of the Zuojin pill was 23.48%. Berberine hydrochloride, Coptis chinensis alkaloid, palmatine, magnolia alkaloid, evodiamine, rutaecarpine, and dehydroevodiamine were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (China). Carbamazepine (internal standard) was purchased from the National Institutes for Food and Drug Control. Berberine hydrochloride, Coptis chinensis alkaloid, palmatine, magnolia alkaloid, evodiamine, rutaecarpine, and dehydroevodiamine were prepared to standard concentrations of 1.1, 5.15, 1.6, 2.55, 12, 17, and 0.5 μg/mL in methanol/water (1 : 1, v : v). Aliquots of all standards and internal standards were stored at −20°C. Linearity was measured using a freshly prepared calibration pool of the mixed standards that was diluted to 1/2, 1/4, 1/10, 1/20, 1/40, 1/100, 1/500, and 1/1000 to obtain 9 final standard concentrations. e final concentration of the internal standard was 10 μg/mL. Each concentration was prepared in three replicates. e calibration curve was prepared by determining the best fit of the peak area ratio (peak area of analyte/internal standard) versus concentration. e calibration curves were constructed using linear regression with 1/x or 1/x2 weighting factors. e results were used to calculate the overall linearity.

HPLC-MS/MS Analysis.
e chromatographic separation was carried out on a Shim-pack GIS C18 column (10 mm × 2.1 mm, 3 μm) fitted on a Jasper HPLC system. e mobile phases were 0.1% formic acid (A) and acetonitrile (B). e flow rate was set to 0.3 mL/min. e column temperature was maintained at 40°C. e following gradient program was used: the initial mixture of 90% A and 10% B was held for 0.5 min; the linear gradient was increased to 70% B in 5 min and held for 2 min; then the linear gradient was increased to 90% B in 0.1 min and held for 1 min; then return to the initial conditions in 0.1 min, followed by 3.3 min of equilibration. e total run time was 12 min, and the injection volume was 2 μL. Mass spectra were obtained using an API 3200 equipped with a TurboIon electrospray (ESI) interface set in the positive mode (needle voltage +5500 V). e mass spectrometry conditions were as follows: CUR, 20 psi; Gas 1 and Gas 2, 55 psi; IS: 5500 V; gas temperature: 550°C. e acquisition dwell time for each transition monitored was 50 ms.

Statistical Analyses.
A series of statistical analyses were conducted by SPSS 26.0. Continuous variables are expressed as 'mean ± standard', and categorical variables are expressed as frequencies (percentages). Student's t-test, Mann-Whitney test, or Chi-square test was used to compare baselines in different circumstances. For the comparison of variations from baseline to endpoint, Student's t-test and Wilcoxon signed-ranks test were performed according to normal or nonnormal variables, respectively. Moreover, the Wilcoxon test was applied to analyze the differences in the endoscopic scores and symptom scores between groups. e Chi-square test or Fisher's exact test was used for the symptom disappearance rate. All statistical tests were twosided. e levels of P < 0.05 and P < 0.01 were assumed to be statistically significant and markedly significant, respectively.

Participant Distribution.
Seventy patients with endoscopy evaluation were determined to be eligible. However, one patient had liver cirrhosis, and one patient refused to sign the informed consent forms. us, a total of 68 patients were eligible for inclusion in this trial according to the CONSORT statement ( Figure 1). All 68 patients were randomly assigned to the control group and treatment group.
irty-four patients were assigned to the treatment group. is group included 22 men and 12 women, and the average age was 56.20 ± 9.33 years.
irty-four patients were included in the control group. is group included 17 men and 17 women, and the average age was 53.91 ± 10.92 years. Two patients were excluded due to loss of follow-up, 1 patient deviated from the protocol, and 1 patient had poor compliance.

Baseline Characteristics.
ere was no significant difference in age, sex, endoscopic scores, histopathological changes, or symptom scores between the two groups. Moreover, the number of patients with H. pylori (positive) in the treatment group (44.12%, 15/34) and that in the control group (52.94%, 18/34) were also comparable (P � 0.355) ( Table 1).

Endoscopy and Histology Evaluation Scores.
Zuojin pill was able to improve the red plaques and the bile reflux scores compared with Marzulene-S granule (P � 0.043 and P � 0.019, respectively). However, there was no significant difference in the erosion score between patients who received the Zuojin pill and the Marzulene-S granule (P � 0.769). Although the Zuojin pill had a trend of decreasing hemorrhage compared with Marzulene-S granule, it did not reach statistical significance (P � 0.405) ( Table 2). In addition, the grade of histopathological changes in chronic inflammation was not significantly different between the two therapies (P � 0.055). However, the Zuojin pill markedly alleviated the active chronic inflammation score compared with the Marzulene-S granule (P � 0.043) ( Table 2).

Subgroup Analysis.
Because Hp infection is thought to be the main cause of CNG, a subgroup analysis based on Hp infection was also conducted. e results indicated that the Zuojin pill could significantly decrease active chronic inflammation compared with Marzulene-S granule treatment in patients with Hp infection (P � 0.033). Moreover, the Zuojin pill was able to alleviate red plaques (P � 0.016), chronic inflammation (P � 0.005) , and abdominal distension (P � 0.012) compared with Marzulene-S granule treatment in patients without Hp infection (Table 3).

Adverse Events.
Adverse events included constipation (1 case in the Zuojin pill group) and dizziness (1 case in the Zuojin pill group and 2 cases in the Marzulene-S granule group). None of the cases were determined to be related to the medication.

e Insight into CNG from Mechanism to Treatment.
CNG is a common digestive disease but has a wide variety of complex mechanisms that require further investigation. e traditional concept of 'one target, one disease' seems inadequate for the determination of this crucial pathogenesis and is a limited approach for treatment [14,15]. erefore, the recent recognition of the pathogenesis of this disease could be beneficial to improving the therapeutic options. It is widely believed that the progression of CNG, CAG, gastric intestinal metaplasia (GIM), and gastric cancer (GC) occurs through a thoroughly programmed deterioration with   Helicobacter pylori is a Gram-negative pathogenic bacterium that has been found to colonize in the gastric mucosa of a large population worldwide. e progress of Helicobacter pylori infection is believed to be the initiating cause of gastritis, and this has been established for almost a century [16]. However, the investigation into its role in inducing inflammatory responses in gastritis has never ended.  Traditional concepts recognize that Helicobacter pylori activates inflammation through various pathways that are associated with modifying TLR ligands [17]. Moreover, the most recent research indicates that Helicobacter pylori could exacerbate gastric inflammation by triggering C-type lectin receptors to modify host cholesterol [18]. Helicobacter pylori infection is widely associated with inflammation and the dysregulation of metabolites, which affects the progression of gastritis to a great extent. Oxidative stress, which plays a double-edged role, has also received increasing attention for its role in the progression of gastritis. First, redox homeostasis and antioxidant defense are highly needed to maintain homeostasis of the gastric epithelium due to excessive exposure to harsh conditions involving digestive enzymes, an aggressive pH environment, and bacteria [19]. For instance, the products of lipid peroxidation, including HNE and its protein/histidine adducts, are thought to be closely related to the pathogenesis of numerous gastric diseases [20]. Interestingly, a beneficial role has emerged for HNE in eradicating Helicobacter pylori infection. A study from Grasberger indicated that the dual oxidases (DUOX) enzyme complex was able to prevent gastric colonization by alleviating Helicobacter pylori and the inflammatory response [21]. In addition, DNA damage and repair are another important cause of chronic gastritis. e p42.3 gene is a typical tumorigenesis promoter in various cancers [22]. It has been proven that both Helicobacter pylori and inflammatory factors can significantly enhance p42.3 expression at the protein level. is gene was also positively associated with the severity of injury to the gastric mucosa. us, the damage caused by p42.3 might be a significant regulator that affects CNG [23]. It is critically important to reverse the complex signaling network associated with CNG progression. e concept of a combination medication for CNG, such as triple therapy, is to maintain the surrounding stability.
is concept has lasted at least 2000 years since the development of traditional Chinese medicine. Zuojin pill is used to treat various gastric diseases that are associated with network regulation. Its gastroprotective effect has been revealed at the molecular level during the past decade. Rhizoma Coptidis, one of the constituents in the Zuojin pill, was able to alleviate chronic gastritis with Helicobacter pylori infection. Our previous pharmacological research revealed that this effect was due to palmatine. It might ameliorate Helicobacter pylori-induced chronic gastritis by inhibiting MMP-10 through the ADAM17/EGFR signaling pathway [24]. Moreover, this action was also related to a metabolite network involving taurine and hypotaurine metabolism, glycerophospholipid metabolism, and pentose and glucuronate interconversions [25]. Another compound, berberine, which is derived from Rhizoma Coptidis, also demonstrates a beneficial effect on chronic gastritis. Research has indicated that berberine is able to suppress the IRF8-IFN-c signaling axis to inhibit progression [26]. Apart from Rhizoma Coptidis, the research on the effects of Fructus Evodiae on gastritis has also vastly improved. Rutaecarpine, one of the main quinazolino carboline alkaloids, exerts a remarkable gastroprotective effect against mucosal injury induced by ethanol. is effect is highly associated with the suppression of the nuclear translocation of NF-κB p65 and its downstream signaling factors. e antioxidant role of this compound also involves the activation of the PI3K/AKT signaling pathway [27].
us, the components of the Zuojin pill are integral to the mechanisms of its effects.

e Guidance of TCM from Bedside to Bench to Bedside.
Establishing an approach that connects basic research and clinical practice has long been an aim in medical science. In the past two decades, translational medicine has been considered a suitable concept for bridging this gap. It acts as a robust bidirectional link between research and application to enhance the rapid clinical translation and provide further feedback for basic research results [28]. During this progress, basic research knowledge can be translated to clinical application. It thus provides more advanced concepts, techniques, tools, and methods to address the diagnosis and treatment of diseases. Moreover, clinical researchers also ruminate the basic advances to further amend deficiencies in basic research. ese relationships are commonly called 'from bench to bedside', 'from bedside to bench', or 'B2B' [29]. ereafter, chemicals were developed for various diseases via this approach [30].
As the cornerstone of traditional medicine, herbal medicine has been recognized as an important source for drug development [31,32]. Moreover, modern basic research offers deep insight into this clinical application and promotes further indication of its use. us, many medicines experience the process of 'from bedside to bench to bedside' to find the optimal guidance in TCM. e anti-influenza virus efficacy of Yinqiao powder (Yinqiaosan) is an outstanding example.
is formula is found in 'Wen Bing TiaoBian', which is the basis of the theoretical research on the warm-heat disease in TCM. It consists of nine herbal components, such as Flos Lonicerae, Fructus forsythiae, Fructus arctii, and Herba Schizonepetae. Yinqiao powder and its modified formulae have a long history in the treatment of the common cold, fever, coughing, and other respiratory infectious diseases. Furthermore, researchers found that Yinqiao powder is able to inhibit the influenza-A virus based on an infected embryonated hen egg model [33]. In 2011, a modified formula of Yinqiao powder demonstrated remarkable efficacy in reducing the time to fever resolution in patients combating H1N1 influenza virus infection in the clinic [8].
is model sheds light on the translational medicine approach for TCM and bridges the gap between clinical experience and medicinal therapy. In addition, our previous research also confirmed the gastroprotective effect of the Zuojin pill and its components against mucosal injury.
us, a clinical trial will provide clinical evidence for the value of the Zuojin pill for further application in CNG.

e Indication of Current Research from Results to Application.
is research mainly focused on the efficacy of the Zuojin pill on CNG in the clinic. e results suggested that the Zuojin pill was able to alleviate several indices of mucosal injury under endoscopy and histology. It could downregulate red plaques and bile reflux but not erosion and hemorrhage, Evidence-Based Complementary and Alternative Medicine indicating the selectivity of Zuojin pill action. In addition, it could remarkably alleviate active chronic inflammation. is action is critically important in anti-inflammation. erefore, this efficacy coincides with our previous experimental results [27]. Furthermore, the efficacy of the Zuojin pill in alleviating symptoms of abdominal distension, belching, and loss of appetite was remarkable, indicating an integral improvement in quality of life. However, this result should be interpreted with caution. e concept of 'pattern' is the core factor for differentiating disease and further treatment in traditional Chinese medicine. at is, the Zuojin pill might be more suitable for CNG patients with a pattern of liver fire invading the stomach in TCM. Patients with this kind of pattern might achieve higher efficacy. us, subgroup trials with different patterns are needed for further exploration. Regarding the Helicobacter pylori positivity rate, the Zuojin pill did not demonstrate a significant difference compared to the Marzulene-S granule.
is is because the main targets of the Zuojin pill focus more on downstream signaling pathways involved in inflammatory regulation. Furthermore, safety was also observed during the whole trial. However, all the cases with adverse events were ultimately determined to have no relation to the medication. erefore, Zuojin pill treatment could be considered safe.

e Limitations of the Current Research.
Overall, this trial strictly abided by the clinical standard from design to the determination to calculation. However, there still exists improvement in three aspects. First, a multicenter randomized clinical trial with large sample size is needed for further exploration. Only by performing this kind of trial can the evidence reported here be applied to guide gastritis treatment. Second, this research mainly uses the modern clinical index of CNG. Zuojin pill alleviates various symptoms. erefore, the index of TCM symptoms should also be evaluated to obtain further clinical data. Finally, a comprehensive investigation of the specific mechanisms is still needed. Due to the current progress of multiomics techniques, the exploration of combined metabolomics and proteomics is urgently needed to predict possible molecular mechanisms and indicate the cure index.

Conclusion
Zuojin pill is able to decrease the mucosal injury of CNG and alleviate the related symptoms to elevate the life of quality. It is also safe for patients to use. In addition, more large-scale clinical trials with longer intervention durations should be carried out to further prove its clinical efficacy and safety.

Data Availability
Data are available in ChiCTR2000040549.

Conflicts of Interest
e authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.