Gut microbiota-derived butyrate restores impaired regulatory T cells in patients with AChR myasthenia gravis via mTOR-mediated autophagy.

More than 80% of patients with myasthenia gravis (MG) are positive for anti-acetylcholine receptor (AChR) antibodies. Regulatory T cells (Tregs) suppress overproduction of these antibodies, and patients with AChR antibody-positive MG (AChR MG) exhibit impaired Treg function and reduced Treg numbers. The gut microbiota and their metabolites play a crucial role in maintaining Treg differentiation and function. However, whether impaired Tregs correlate with gut microbiota activity in patients with AChR MG remains unknown. Here, we demonstrate that butyric acid-producing gut bacteria and serum butyric acid level are reduced in patients with AChR MG. Butyrate supplementation effectively enhanced Treg differentiation and their suppressive function of AChR MG. Mechanistically, butyrate activates autophagy of Treg cells by inhibiting the mammalian target of rapamycin. Activation of autophagy increased oxidative phosphorylation and surface expression of cytotoxic T-lymphocyte-associated protein 4 on Treg cells, thereby promoting Treg differentiation and their suppressive function in AChR MG. This observed effect of butyrate was blocked using chloroquine, an autophagy inhibitor, suggesting the vital role of butyrate-activated autophagy in Tregs of patients with AChR MG. We propose that gut bacteria derived butyrate has potential therapeutic efficacy against AChR MG by restoring impaired Tregs.

Association of Antidiabetic Drug Target Genes with Inflammatory Bowel Disease: A Mendelian Randomization Study.

Background: An unmet medical need for the treatment of inflammatory bowel disease (IBD) exists. A part of antidiabetic drugs had potential effects on IBD in various observational research. Objective: To investigate the potential of antidiabetic drugs on IBD. Methods: We undertook a summary-data-based Mendelian randomization (SMR) using the expression quantitative trait loci (eQTL) expressed in the blood or colon and a two sample Mendelian randomization (TSMR) utilizing single nucleotide polymorphism (SNP) of antidiabetic drug target genes mediated by blood glucose traits. Participants encompassed patients with IBD (25,042 cases/34,915 controls), UC (12,366 cases/33,609 controls), and CD (12,194 cases/28,072 controls). Data on eQTL in the blood or the colon were from the eQTLGen consortium (31,684 individuals) or GTEx Consortium V8, respectively. SMR was performed by SMR software (20,220,322); the primary method for TSMR was inverse-variance weighted (IVW) or Wald ratio through R studio (2023.06.0+421). Sensitivity analyses were carried out. Results: A 1-SD upper expression of the KCNJ11 gene (target gene of sulfonylureas) in the blood reduced the risk of CD (OR per 1-SD = 0.728, 95% CI = 0.586-0.903, P = 0.004) according to the result of SMR. ABCC8 (target gene of sulfonylureas) expressed in the colon did not affect CD, UC, or IBD. T2D-mediated KCNJ11 has a protective effect on CD (OR = 0.475, 95% CI = 0.297-0.761, P = 0.002). Gene predicted no relationship between T2D and CD. Conclusion: Sulfonylureas (SUs) may have side effects on CD. This work provides some suggestions for the selection of antidiabetic drugs in patients with CD.

Causal Association Analysis of Periodontitis and Inflammatory Bowel Disease: A Bidirectional Mendelian Randomization Study.

BACKGROUND: Periodontitis has been reported to be associated with inflammatory bowel disease (IBD), including ulcerative colitis (UC), and Crohn's disease (CD). However, the causality of these 2 diseases remains unclear. We conducted bidirectional Mendelian randomization (MR) to investigate the causal relationship between periodontitis and IBD. METHODS: We obtained the genome-wide association study (GWAS) summary data of European populations from FinnGen database (for IBD) and a published article (for periodontitis), from which independent single nucleotide polymorphisms were selected as instrumental variables. Inverse variance-weighted (IVW), MR-Egger, and weighted median (WM) methods were utilized for MR analysis. Heterogeneity or pleiotropy was detected through Cochran's Q test and MR-Egger intercept, respectively. Outlier was identified with MR-PRESSO (Mendelian Randomization Pleiotropy RESidual Sum and Outlier) and leave-one-out analysis. All statistical analyses were performed with R 4.2.1 and the packages of TwoSampleMR version 0.5.6. RESULTS: Genetic prediction showed that periodontitis was the risk factor of UC (odds ratio [OR], 1.13; 95% confidence interval [CI], 1.01-1.26; P = .027), rather than of CD (OR, 0.92; 95% CI, 0.74-1.15; P = .456) and IBD (OR, 0.96; 95% CI, 0.81-1.13; P = .619). To the contrary, CD, not UC or IBD, resulted in exacerbating periodontitis in terms of the results of the IVW (OR, 1.09; 95% CI, 1.01-1.17; P = .021) and WM (OR, 1.10; 95% CI, 1.01-1.20; P = .030) methods. Heterogeneity or pleiotropy was acceptable. CONCLUSIONS: Our results indicated that CD was the risk factor for periodontitis; conversely, periodontitis was responsible for the exacerbation of UC, enhancing the existence of mouth-gut axis. Patients with UC should pay more attention to periodontal health, while patients with periodontitis should actively pay close heed to intestinal health.

A bidirectional Mendelian randomization study indicated that Crohn's disease was the risk factor for periodontitis; conversely, periodontitis was responsible for the exacerbation of ulcerative colitis, enhancing the existence of the mouth-gut axis and suggesting paying attention to oral health for patients of inflammatory bowel disease.

Symptom effects and central mechanism of acupuncture in patients with functional gastrointestinal disorders: a systematic review based on fMRI studies.

BACKGROUND: Functional gastrointestinal disorders (FGIDs) are closely related to disorders of brain-gut interaction. FGIDs are the dominant disease of acupuncture treatment, which can improve the symptoms and emotional state. AIM: To evaluate the results and quality of the available clinical evidence and to summarize the central mechanism and effect of acupuncture on FGIDs. METHODS: PubMed, EMBASE, Web of science, Cochrane Library, China National Knowledge Infrastructure (CNKI) were searched by computer to collect the randomized controlled trials (RCTs), which contained central mechanisms via fMRI research of acupuncture in the treatment of FGIDs patients. The search time limit was from the establishment of the database to June 22, 2022. Two researchers independently screened the literature, extracted data, and evaluated the quality. RESULTS: Ten RCTs involving fMRI data were included in this study, including 4 Functional dyspepsia (FD) studies, 3 irritable bowel syndrome (IBS) studies, and 3 functional constipation (FC) studies. The score of improvements in both gastrointestinal symptoms and psychological symptoms showed that acupuncture could significantly improve the clinical symptoms of FGIDs patients, including abdominal pain, abdominal distension, frequency of defecation, and stool characteristics, and could relieve anxiety and depression symptoms of patients. Acupuncture could regulate brain functional connections and functional activity in FGIDs patients, mainly including insula, anterior cingulate cortex, prefrontal cortex, thalamus, hippocampus, amygdala and other brain regions. CONCLUSION: Acupuncture can improve gastrointestinal symptoms and psychological status in FGIDs patients, and regulate functional connectivity and activity of brain regions such as insula, ACC, PFC, thalamus, HIPP, amygdala, etc. These changes in brain activity may related to visceral sensation, pain regulation, emotion, but further studies of high quality are still necessary.

Astragaloside IV ameliorates experimental autoimmune myasthenia gravis by regulating CD4 + T cells and altering gut microbiota.

BACKGROUND: Myasthenia gravis (MG) is an antibody-mediated autoimmune disease and its pathogenesis is closely related to CD4 + T cells. In recent years, gut microbiota is considered to play an important role in the pathogenesis of MG. Astragaloside IV (AS-IV) is one of the main active components extracted from Astragalus membranaceus and has immunomodulatory effects. To study the immunomodulatory effect of AS-IV and the changes of gut microbiota on experimental autoimmune myasthenia gravis (EAMG) mice, we explore the possible mechanism of AS-IV in improving MG. METHODS: In this study, network pharmacology was utilized to screen the crucial targets of AS-IV in the treatment of MG. Subsequently, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify potential pathways through which AS-IV acts against MG. Furthermore, experimental investigations were conducted to validate the underlying mechanism of AS-IV in MG treatment. Before modeling, 5 mice were randomly selected as the control group (CFA group), and the other 10 were induced to EAMG model. These mice were randomly divided into EAMG group and EAMG + AS-IV group, n = 5/group. In EAMG + AS-IV group, AS-IV was administered by gavage. CFA and EAMG groups were given the same volume of PBS. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. At the last administration, the feces were collected for 16S RNA microbiota analysis. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected by flow cytometry. The levels of IFN-γ, IL-17 and TGF-ß in serum were measured by ELISA. Furthermore, fecal microbial transplantation (FMT) experiments were performed for exploring the influence of changed intestinal flora on EAMG. After EAMG model was induced, the mice were treated with antibiotics daily for 4 weeks to germ-free. Then germ-free EAMG mice were randomly divided into two groups: FMT EAMG group, FMT AS-IV group, n = 3/group. Fecal extractions from EAMG and EAMG + AS-IV groups as gathered above were used to administered daily to the respective groups for 4 weeks. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected at the last administration. The levels of IFN-γ, IL-17 and TGF-ß in serum were measured by ELISA. RESULTS: The network pharmacology and KEGG pathway analysis revealed that AS-IV regulates T cell pathways, including T cell receptor signaling pathway and Th17 cell differentiation, suggesting its potential in improving MG. Further experimental verification demonstrated that AS-IV administration improved muscle strength and body weight, reduced the level of Th1 and Th17 cells, enhanced the level of Treg cells, and resulted in alterations of the gut microbiota, including changes in beta diversity, the Firmicutes/Bacteroidetes (F/B) ratio, and the abundance of Clostridia in EAMG mice. We further conducted FMT tests and demonstrated that the EAMG Abx-treated mice which were transplanted the feces of mice treated with AS-IV significantly alleviated myasthenia symptoms, reduced Th1 and Th17 cells levels, and increased Treg cell levels. CONCLUSION: This study speculated that AS-IV ameliorates EAMG by regulating CD4 + T cells and altering the structure and species of gut microbiota of EAMG.

Paris polyphylla extract attenuates colitis in mice by regulating PPAR-γ mediated Treg/Th17 balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Paris polyphylla Sm. (P.P), is a widely-used traditional Chinese medicine (TCM) in the treatment of wound, throat sores and snakebites. Furthermore, P.P was recorded as an anti-inflammatory drug by the Chinese Pharmacopoeia. AIM OF THE STUDY: We sought to decipher the anti-inflammatory effect of P.P on ulcerative colitis (UC); specifically, to explore whether P.P attenuates colitis by restoring the regulatory T cells (Tregs) and T helper 17 (Th17) cells balance and its mechanism. MATERIAL AND METHODS: We treated experimental colitis mice with extracts of Paris polyphylla (EPP). The percentage of Tregs and Th17 cells were measured using flow cytometry, and their secreted cytokines levels were evaluated employing ELISA. The expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) in colon tissues was detected using immunofluorescence. Furthermore, GW9662, a PPAR-γ antagonist, was used to validate the mechanism of EPP in restoring the Treg/Th17 balance. RESULTS: The EPP effectively alleviated the clinical symptoms and inflammatory cytokine levels in mice with colitis. EPP treatment also restored the impaired Treg/Th17 balance in mice. Furthermore, EPP treatment promoted PPAR-γ expression and reduced HIF-1α and p-STAT3 expression in colon tissues, whereas PPAR-γ inhibition blocked the effects of EPP in mice models. CONCLUSION: Our study indicates that EPP exhibit excellent anti-inflammatory properties via restoring PPAR-γ/STAT3/HIF-1α axis-mediated Treg/Th17 balance in colitis mice. Hence, P. polyphylla is a promising medicinal plant-based alternative for managing colitis that requires further clinical validation.

Effectiveness of modified Buzhong Yiqi decoction in treating myasthenia gravis: study protocol for a series of N-of-1 trials.

BACKGROUND: Myasthenia gravis (MG) is an acquired autoimmune disease with high heterogeneity. The disease is chronic, relapsing repeatedly and progressive with acute exacerbation occasionally. Although the treatment of MG has developed, it is still unsatisfactory and has some unexpected side effects. Traditional Chinese medicine (TCM) has shown great potential in MG treatment, including relief of muscle weakness syndrome, improvement of patient's quality of life, and reduction of side effects of western medicine. The purpose of this study is to evaluate the effectiveness of modified Buzhong Yiqi decoction (MBYD) as an add-on therapy for MG through a small series of N-of-1 trials. METHODS: Single-centre, randomized, double-blind, 3 crossover N-of-1 trials will be conducted to enroll patients with MG diagnosed as spleen-stomach deficiency syndrome or spleen-kidney deficiency syndrome in TCM. Each N-of-1 trial has 3 cycles of two 4-week periods containing the MBYD period and placebo period. The wash-out interval of 1 week is prior to switching each period. PRIMARY OUTCOME: quantitative myasthenia gravis (QMG). SECONDARY OUTCOMES: the following scales: myasthenia gravis composite (MGC), myasthenia gravis activities of daily living profile (MG-ADL), myasthenia gravis quality of life (MG-QOL); the level of CD4+FoxP3+Treg cells and cytokines (IL-4, IL-17A, INF-γ, TGF-ß) in the peripheral blood; the alterations of the composition of gut microbiota; reduction of the side effects of western medicine. DISCUSSION: Used by WinBUGS software, we will conduct a hierarchical Bayesian statistical method to analyze the efficacy of MBYD in treating MG in individuals and populations. Some confounding variables such as TCM syndrome type and potential carryover effect of TCM will be introduced into the hierarchical Bayesian statistical method to improve the sensitivity and applicability of the trials, and the use of prior available information within the analysis may improve the sensitivity of the results of a series of N-of-1 trials, from both the individual and population level to study the efficacy of TCM syndrome differentiation. We assumed that this study would reveal that MBYD is effective for MG and provide robust evidence of the efficacy of TCM to treat MG. TRIAL REGISTRATION: Chinese Clinical Trial Register, ID: ChiCTR2000040477 , registration on 29 November 2020.

Stigmasterol Restores the Balance of Treg/Th17 Cells by Activating the Butyrate-PPARγ Axis in Colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with gut microbiota disequilibrium and regulatory T (Treg)/T helper 17 (Th17) immune imbalance. Stigmasterol, a plant-derived sterol, has shown anti-inflammatory effects. Our study aimed to identify the effects of stigmasterol on experimental colitis and the related mechanisms. Stigmasterol treatment restored the Treg/Th17 balance and altered the gut microbiota in a dextran sodium sulfate (DSS)-induced colitis model. Transplantation of the faecal microbiota of stigmasterol-treated mice significantly alleviated inflammation. Additionally, stigmasterol treatment enhanced the production of gut microbiota-derived short-chain fatty acids (SCFAs), particularly butyrate. Next, human naïve CD4+ T cells sorted from IBD patients were cultured under Treg- or Th17-polarizing conditions; butyrate supplementation increased the differentiation of Tregs and decreased Th17 cell differentiation. Mechanistically, butyrate activated peroxisome proliferator-activated receptor gamma (PPARγ) and reprogrammed energy metabolism, thereby promoting Treg differentiation and inhibiting Th17 differentiation. Our results demonstrate that butyrate-mediated PPARγ activation restores the balance of Treg/Th17 cells, and this may be a possible mechanism, by which stigmasterol attenuates IBD.

The Synergistic Effects of 5-Aminosalicylic Acid and Vorinostat in the Treatment of Ulcerative Colitis.

Background: The drug 5-aminosalicylic acid (5-ASA) is the first-line therapy for the treatment of patients with mild-to-moderate ulcerative colitis (UC). However, in some cases, 5-ASA cannot achieve the desired therapeutic effects. Therefore, patients have to undergo therapies that include corticosteroids, monoclonal antibodies or immunosuppressants, which are expensive and may be accompanied by significant side effects. Synergistic drug combinations can achieve greater therapeutic effects than individual drugs while contributing to combating drug resistance and lessening toxic side effects. Thus, in this study, we sought to identify synergistic drugs that can act synergistically with 5-ASA. Methods: We started our study with protein-metabolite analysis based on peroxisome proliferator-activated receptor gamma (PPARG), the therapeutic target of 5-ASA, to identify more additional potential drug targets. Then, we further evaluated the possibility of their synergy with PPARG by integrating Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis, pathway-pathway interaction analysis, and semantic similarity analysis. Finally, we validated the synergistic effects with in vitro and in vivo experiments. Results: The combination of 5-ASA and vorinostat (SAHA) showed lower toxicity and mRNA expression of p65 in human colonic epithelial cell lines (Caco-2 and HCT-116), and more efficiently alleviated the symptoms of dextran sulfate sodium (DSS)-induced colitis than treatment with 5-ASA and SAHA alone. Conclusion: SAHA can exert effective synergistic effects with 5-ASA in the treatment of UC. One possible mechanism of synergism may be synergistic inhibition of the nuclear factor kappa B (NF-kB) signaling pathway. Moreover, the metabolite-butyric acid may be involved.

Network pharmacology dissection of multiscale mechanisms for jiaoqi powder in treating ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence of ulcerative colitis (UC) is increasing worldwide, making it a serious public health challenge. Currently, there are no accepted curative treatments for UC. As such, the exploration of new therapeutic strategies for UC treatment is of considerable clinical importance. Jiaoqi powder (JQP) is a classic Chinese medicinal formula commonly used as a complementary and alternative medicine for treating gastrointestinal bleeding. JQP is thus a potential alternative medicine for UC treatment. However, the protective mechanism underlying the action of JQP has not been elucidated, thereby, necessitating further studies to decipher the mechanisms involved in the complex interplay among its components. AIM OF THE STUDY: To explore the protective effect of JQP against UC and to further investigate its mechanism in silico and in vivo using a systems pharmacology approach. MATERIALS AND METHODS: A systems pharmacology approach was used to predict the active components of JQP. Putative targets and the potential mechanism of JQP on UC were obtained through target fishing, network construction, and enrichment analyses. An animal-based model of dextran sodium sulfate (DSS)-induced colitis in C57BL/6 mice was further used to validate the treatment mechanisms of JQP. The underlying pharmacological mechanisms of JQP in UC were determined using polymerase chain reaction tests, histological staining, immunohistochemistry, enzyme-linked immunoassays, and flow cytometry analysis. RESULTS: In this study, 17 effective components and 941 potential targets of JQP were identified. Similarly, 2104 UC-related targets were also identified. Construction of PPI networks led to the identification of 184 putative therapeutic targets of JQP. Sixty-nine core targets among these 184 were further screened based on their DC values. Gene ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the core targets were primarily enriched in immune response and inflammatory signalling pathways. Subsequent animal-based in vivo experiments revealed that JQP ameliorated symptoms and histological changes in DSS colitis by significantly impairing DSS's ability to induce high expression levels of NF-κB/p65, IL-1ß, IL-6, and TNF-α. JQP also reduced the levels of COX-2, CCL2, CXCL2, HIF-1α, MMP3 and MMP9 and regulated the Th17/Treg cell balance in DSS-induced mice. CONCLUSIONS: This study demonstrated that JQP could treat UC by improving the mucosal inflammatory response, repairing the intestinal barrier, and modulating the Th17/Treg immune balance. The results of this study provide new insights into UC treatment and further elucidate the theoretical and practical implications of the pharmaceutical development of TCMs.

Prognostic Model of Colorectal Cancer Constructed by Eight Immune-Related Genes.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumor of the digestive tract with a high mortality rate. Growing evidence demonstrates that immune-related genes play a prominent role in the occurrence and development of CRC. The aim of this study was to investigate the prognostic value of immune-related genes in CRC. METHODS: Gene expression profiles and clinical data of 568 CRC and 44 non-tumorous tissues were obtained from The Cancer Genome Atlas (TCGA) database. First, we performed a differentially expressed gene (DEG) analysis and univariate Cox regression analysis to determine the DEGs associated with overall survival. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently performed for prognostic immune-related genes. Then, a multivariate Cox regression analysis was performed to establish the immune prognostic model and identify the independent prognostic factors of CRC. Next, in vitro experiments were done to further validate the model. Finally, we analyzed the correlation among immune-related genes, clinical traits, and immune cell infiltration. RESULTS: In total, 3,702 DEGs were obtained, and 338 prognostic immune-related genes were identified. Among them, 45 genes were significantly correlated with the prognosis of CRC patients. A TF-mediated network was set up to explore its internal mechanism. GO and KEGG analyses further illustrated that these genes were enriched in immune-and inflammatory-related pathways. Then, a prognostic prediction model composed of eight immune-related genes (SLC10A2, UTS2, FGF2, UCN, IL1RL2, ESM1, ADIPOQ, and VIP) was constructed. The AUC of the ROC curve for 1, 3, 5, and 10 years overall survival (OS) was 0.751, 0.707, 0.680, and 0.729, respectively. The survival analysis suggested that the OS of the high-risk group was significantly poorer than that of the low-risk group. Meanwhile, in vitro assays revealed that ESM1 and SLC10A2 exert opposing roles in colon cancer cell proliferation, validating the accuracy of the model. The correlation analysis indicated that immune cell infiltration was positively related to the model. CONCLUSION: This study screened prognosis-related immune genes and developed a prognostic prediction model of CRC. These findings may help provide potential novel prognostic biomarkers and therapeutic targets for CRC. At the same time, the understanding of the CRC immune microenvironment status was deepened.

Therapeutic efficacy and immunoregulatory effect of Qiangji Jianli Capsule for patients with myasthenia gravis: Study protocol for a series of randomized, controlled N-of-1 trials.

INTRODUCTION: Myasthenia gravis (MG) is an autoimmune disease in which antibodies directly target components of the neuromuscular junction, causing neuromuscular conduction damage that leads to muscle weakness. The current pharmaceutical treatment for MG is still not ideal to address the problems of disease progression, high recurrence rate, and drug side effects. Clinical observations suggest that traditional Chinese medicine (TCM) can strengthen immunity and improve symptoms of MG patients, delay the progression of the disease, reduce or even prevent the need for immunosuppressive therapy when used in combination with acetylcholinesterase inhibitors or low-dose prednisone, as well as improve the quality of life of patients. The Qiangji Jianli Capsule (QJC) is a combination of medicinal herbs which is used in traditional Chinese medicine. Since MG is a rare disorder, randomized controlled trials comparing large cohorts are difficult to conduct. Therefore, we proposed to aggregate data from a small series of N-of-1 trials to assess the effect of the Chinese medical prescription QJC, which strengthens the spleen and nourishes Qi, as an add-on treatment for MG with spleen and stomach Qi deficiency syndrome. METHODS AND ANALYSIS: Single-center, randomized, double-blind, multiple crossover N-of-1 studies will compare QJC versus placebo in 5 adult MG patients with spleen and stomach Qi deficiency syndrome. Patients will undergo 3 cycles of two 4-week intervention periods. According to the treatment schedule, patients will continue to be treated with pyridine bromide tablets, prednisone acetate, tablets and/or tacrolimus capsules throughout the entire trial. Each period consisting of 4-week oral add-on treatment with QJC will be compared with 4-week add-on treatment with a placebo. The primary endpoints are quantitative myasthenia gravis (QMG) test; measurement of the amount of Treg cells and cytokines such as interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-17A (IL-17A), and transforming growth factor-ß (TGF-ß); and corticosteroid or immunosuppressive agent dosage. Secondary outcome measures: Clinical: Evaluation of the effect of TCM syndromes; MG-activities of daily living (MG-ADL) scales; adverse events. ETHICS AND DISSEMINATION: This study was approved by The First Affiliated Hospital of Guangzhou University of Chinese Medicine (GZUCM), No. ZYYECK[2019]038. The results will be published in a peer-reviewed publication. Regulatory stakeholders will comment on the suitability of the trial for market authorization and reimbursement purposes. Trial registration: Chinese Clinical Trial Register, ID: ChiCTR2000033516. Registered on 3 June 2020, http://www.chictr.org.cn/showprojen.aspx?proj=54618.