Insights into the role of RNA m6A modification in the metabolic process and related diseases.

According to the latest consensus, many traditional diseases are considered metabolic diseases, such as cancer, type 2 diabetes, obesity, and cardiovascular disease. Currently, metabolic diseases are increasingly prevalent because of the ever-improving living standards and have become the leading threat to human health. Multiple therapy methods have been applied to treat these diseases, which improves the quality of life of many patients, but the overall effect is still unsatisfactory. Therefore, intensive research on the metabolic process and the pathogenesis of metabolic diseases is imperative. N6-methyladenosine (m6A) is an important modification of eukaryotic RNAs. It is a critical regulator of gene expression that is involved in different cellular functions and physiological processes. Many studies have indicated that m6A modification regulates the development of many metabolic processes and metabolic diseases. In this review, we summarized recent studies on the role of m6A modification in different metabolic processes and metabolic diseases. Additionally, we highlighted the potential m6A-targeted therapy for metabolic diseases, expecting to facilitate m6A-targeted strategies in the treatment of metabolic diseases.

Plasma and urine proteomics and gut microbiota analysis reveal potential factors affecting COVID-19 vaccination response.

The efficacy of COVID-19 vaccination relies on the induction of neutralizing antibodies, which can vary among vaccine recipients. In this study, we investigated the potential factors affecting the neutralizing antibody response by combining plasma and urine proteomics and gut microbiota analysis. We found that activation of the LXR/FXR pathway in plasma was associated with the production of ACE2-RBD-inhibiting antibodies, while urine proteins related to complement system, acute phase response signaling, LXR/FXR, and STAT3 pathways were correlated with neutralizing antibody production. Moreover, we observed a correlation between the gut microbiota and plasma and urine proteins, as well as the vaccination response. Based on the above data, we built a predictive model for vaccination response (AUC = 0.85). Our study provides insights into characteristic plasma and urine proteins and gut microbiota associated with the ACE2-RBD-inhibiting antibodies, which could benefit our understanding of the host response to COVID-19 vaccination.

PROTAC-mediated NR4A1 degradation as a novel strategy for cancer immunotherapy.

An effective cancer therapy requires killing cancer cells and targeting the tumor microenvironment (TME). Searching for molecules critical for multiple cell types in the TME, we identified NR4A1 as one such molecule that can maintain the immune suppressive TME. Here, we establish NR4A1 as a valid target for cancer immunotherapy and describe a first-of-its-kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 degrades NR4A1 within hours in vitro and exhibits long-lasting NR4A1 degradation in tumors with an excellent safety profile. NR-V04 inhibits and frequently eradicates established tumors. At the mechanistic level, NR-V04 induces the tumor-infiltrating (TI) B cells and effector memory CD8+ T (Tem) cells and reduces monocytic myeloid-derived suppressor cells (m-MDSC), all of which are known to be clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anticancer immune responses and offers a new avenue for treating various types of cancers such as melanoma.

Modification patterns and metabolic characteristics of m6A regulators in digestive tract tumors.

M6A is essential for tumor occurrence and progression. The expression patterns of m6A regulators differ in various kinds of tumors. Transcriptomic expression statistics together with clinical data from a database were analyzed to distinguish patients with digestive tract tumors. Based on the expression patterns of diverse m6A regulators, patients were divided into several clusters. Survival analysis suggested significant differences in patient prognosis among the m6A clusters. The results showed overlapping of m6A expression patterns with energy metabolism and nucleotide metabolism. Functional analyses imply that m6A modifications in tumor cells probably drive metabolic reprogramming to sustain rapid proliferation of cancer cells. Our analysis highlights the m6A risk characterizes various kinds of metabolic features and predicts chemotherapy sensitivity in digestive tract tumors, providing evidence for m6A regulators as markers to predict patient outcomes.

Comparative chloroplast genome analyses provide insights into evolutionary history of Rhizophoraceae mangroves.

Background: The Rhizophoraceae family comprises crucial mangrove plants that inhabit intertidal environments. In China, eight Rhizophoraceae mangrove species exist. Although complete chloroplast (Cp) genomes of four Rhizophoraceae mangrove plants have been reported, the Cp genomes of the remaining four species remain unclear, impeding a comprehensive understanding of the evolutionary history of this family. Methods: Illumina high-throughput sequencing was employed to obtain the DNA sequences of Rhizophoraceae species. Cp genomes were assembled by NOVOPlasty and annotated using CpGAVAS software. Phylogenetic and divergence time analyses were conducted using MEGA and BEAST 2 software. Results: Four novel Cp genomes of Rhizophoraceae mangrove species (Bruguiera sexangula, Bruguiera gymnorrhiza, Bruguiera × rhynchopetala and Rhizophora apiculata) were successfully assembled. The four Cp genomes ranged in length from 163,310 to 164,560 bp, with gene numbers varying from 124 to 128. The average nucleotide diversity (Pi) value of the eight Rhizophoraceae Cp genomes was 0.00596. Phylogenetic trees constructed based on the complete Cp genomes supported the monophyletic origin of Rhizophoraceae. Divergence time estimation based on the Cp genomes of representative species from Malpighiales showed that the origin of Rhizophoraceae occurred at approximately 58.54-50.02 million years ago (Mya). The divergence time within the genus Rhizophora (∼4.51 Mya) was much earlier than the divergence time within the genus Bruguiera (∼1.41 Mya), suggesting recent speciation processes in these genera. Our data provides new insights into phylogenetic relationship and evolutionary history of Rhizophoraceae mangrove plants.

Antifungal activity of Klebsiella grimontii DR11 against Fusarium oxysporum causing soybean root rot.

AIMS: Soybean root rot, caused by Fusarium oxysporum, leads to significant economic and financial losses to the soybean processing industry globally. In the study, we aimed to explore a biocontrol agent to combat F. oxysporum infection in soybean. METHODS AND RESULTS: From soybean rhizosphere soil, 48 strains were isolated. Among them, the strain DR11 exhibited the highest inhibition rate of 72.27%. Morphological, physiological, biochemical, and 16S rDNA identification revealed that the strain DR11 was Klebsiella grimontii DR11. Strain DR11 could inhibit the growth of F. oxysporum and spore formation and alter the mycelial morphology. At 5.0 × 106 CFU mL-1, pH 7, and 30°C, it exhibited the highest inhibitory rate (72.27%). Moreover, it could decrease the activity of cell-wall-degrading enzymes of F. oxysporum. Simultaneously, the activities of defense-related enzymes and content of malondialdehyde in soybean plants were increased after treatment with strain DR11. In addition, strain DR11 could form aggregates to form biofilm and adsorb on the surface of soybean roots. It inhibited F. oxysporum growth on soybean seedlings, with an inhibitory effect of 62.71%. CONCLUSION: Klebsiella grimontii DR11 had a strong inhibitory effect on F. oxysporum and could be used as a biocontrol agent to combat F. oxysporum infection in soybean.

Root microbiota alters response to root rot in Rhododendron delavayi Franch.

Root microbiota have a significant effect on plant health. However, the role of root microbiota in the resistance of Rhododendron against root rot is not known. In this study, we employed amplicon 16S and ITS sequencing to investigate the bacterial and fungal communities associated with four distinct niches (bulk soil, rhizosphere, rhizoplane, and endosphere) of both healthy and diseased Rhododendron plants in the Baili Rhododendron nature reserve in China. The amplicon data analysis identified 182 bacterial genera and 141 fungal genera that were impacted by root rot across all niches. Specifically, the rhizoplane appeared to exert a selective gating effect, resulting in a reduction in the complexity of bacterial communities, but not fungal communities, in wild Rhododendron delavayi Franch roots. Nevertheless, the stress induced by root rot led to alterations in the root microbiota and compromised the gating function of the rhizoplane, thereby significantly increasing the complexity of the bacterial community within the plant root. In the root tissue following root rot outbreak, the relative abundance of the pathogenic species Pezicula brunnea and Diaporthe helianthi was enriched by as much as 6.13% and 1.71%, respectively. These findings provide novel insights into the contribution of enrichment of root-associated microbiota to wild plant hosts under the disease stress of root rot. The root rot-causing pathogenic fungi may interact with beneficial bacteria and induce plants to send out "cry for help" signals, which may encourage the specific assembly of microbiota. In the Rhododendron delavayi Franch root microbiota, we found 23 potentially beneficial microbes. Notably, certain beneficial bacteria, such as Sporolactobacillus and Stenotrophomonas, were found to accumulate in the rhizoplane and endosphere under root rot disease stress. Overall, our results lend support to our hypothesis that Rhododendron recruits protective microbes as a strategy to suppress root rot outbreaks. Future endeavors in isolating beneficial microbes capable of mitigating root rot have the potential to enhance plant resilience against root diseases.

Unleashing the Power of NR4A1 Degradation as a Novel Strategy for Cancer Immunotherapy.

An effective cancer therapy requires both killing cancer cells and targeting tumor-promoting pathways or cell populations within the tumor microenvironment (TME). We purposely search for molecules that are critical for multiple tumor-promoting cell types and identified nuclear receptor subfamily 4 group A member 1 (NR4A1) as one such molecule. NR4A1 has been shown to promote the aggressiveness of cancer cells and maintain the immune suppressive TME. Using genetic and pharmacological approaches, we establish NR4A1 as a valid therapeutic target for cancer therapy. Importantly, we have developed the first-of-its kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 effectively degrades NR4A1 within hours of treatment in vitro and sustains for at least 4 days in vivo, exhibiting long-lasting NR4A1-degradation in tumors and an excellent safety profile. NR-V04 leads to robust tumor inhibition and sometimes eradication of established melanoma tumors. At the mechanistic level, we have identified an unexpected novel mechanism via significant induction of tumor-infiltrating (TI) B cells as well as an inhibition of monocytic myeloid derived suppressor cells (m-MDSC), two clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anti-cancer immune responses and offers a new avenue for treating various types of cancer.

HDAC3 and HDAC8 PROTAC dual degrader reveals roles of histone acetylation in gene regulation.

HDAC3 and HDAC8 have critical biological functions and represent highly sought-after therapeutic targets. Because histone deacetylases (HDACs) have a very conserved catalytic domain, developing isozyme-selective inhibitors remains challenging. HDAC3/8 also have deacetylase-independent activity, which cannot be blocked by conventional enzymatic inhibitors. Proteolysis-targeting chimeras (PROTACs) can selectively degrade a target enzyme, abolishing both enzymatic and scaffolding function. Here, we report a novel HDAC3/8 dual degrader YX968 that induces highly potent, rapid, and selective degradation of both HDAC3/8 without triggering pan-HDAC inhibitory effects. Unbiased quantitative proteomic experiments confirmed its high selectivity. HDAC3/8 degradation by YX968 does not induce histone hyperacetylation and broad transcriptomic perturbation. Thus, histone hyperacetylation may be a major factor for altering transcription. YX968 promotes apoptosis and kills cancer cells with a high potency in vitro. YX968 thus represents a new probe for dissecting the complex biological functions of HDAC3/8.

Water Surface Targets Detection Based on the Fusion of Vision and LiDAR.

The use of vision for the recognition of water targets is easily influenced by reflections and ripples, resulting in misidentification. This paper proposed a detection method based on the fusion of 3D point clouds and visual information to detect and locate water surface targets. The point clouds help to reduce the impact of ripples and reflections, and the recognition accuracy is enhanced by visual information. This method consists of three steps: Firstly, the water surface target is detected using the CornerNet-Lite network, and then the candidate target box and camera detection confidence are determined. Secondly, the 3D point cloud is projected onto the two-dimensional pixel plane, and the confidence of LiDAR detection is calculated based on the ratio between the projected area of the point clouds and the pixel area of the bounding box. The target confidence is calculated with the camera detection and LiDAR detection confidence, and the water surface target is determined by combining the detection thresholds. Finally, the bounding box is used to determine the 3D point clouds of the target and estimate its 3D coordinates. The experiment results showed this method reduced the misidentification rate and had 15.5% higher accuracy compared with traditional CornerNet-Lite network. By combining the depth information from LiDAR, the position of the target relative to the detection coordinate system origin could be accurately estimated.

Facile Synthesis of Tetraphenylethene (TPE)-Based Fluorophores Derived by π-Extended Systems: Opposite Mechanofluorochromism, Anti-Counterfeiting and Bioimaging.

Although remarkable progresses are achieved in the design and development of the mono-shift in photoluminescence for mechanofluorochromic materials, it is still a severe challenge to explore the opposite mechanofluorochromic materials with both blue- and red-shifted photoluminescence. Herein, two unprecedented 4,5-bis(TPE)-1H-imidazole fused pyridine or quinoline-based fluorophores X-1 and X-2 were designed and synthesized, and X-1 and X-2, exhibit completely opposite mechanofluorochromic behavior. Under UV lamp, the color of pristine X-1 changed from blue to green with reversible redshifted 27 nm in fluorescence emission spectra after ground, while the color of pristine X-2 changed from red to yellow with reversible blue-shifted 74 nm after ground. The detailed characterizations (including PXRD, SEM and DSC) confirmed that this opposite mechanofluorochromism was attributed to the transformation of order-crystalline and amorphous states. The crystal structure analysis and theoretical calculation further explain that opposite mechanofluorochromic behavior take into account different π-π stacking mode by induced π-extended systems. In addition, these TPE-based fluorophores (X-1 and X-2) exhibited excellent bio-compatibility and fluorescence properties for bio-imaging, writable data storage and anti-counterfeiting materials.

Piperlongumine conjugates induce targeted protein degradation.

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.

The hTERT-p50 homodimer inhibits PLEKHA7 expression to promote gastric cancer invasion and metastasis.

Although accumulating evidence has highlighted the molecular mechanisms by which hTERT promotes tumour cell invasion and metastasis, the molecular mechanisms of the properties enabling hTERT to contribute to invasion and metastasis have not been clearly illustrated. Here, we report that hTERT promotes gastric cancer invasion and metastasis by recruiting p50 to synergistically inhibit PLEKHA7 expression. We observed that the expression of PLEKHA7 in gastric cancer was significantly negatively associated with the TNM stage and lymphatic metastasis and that decreased PLEKHA7 expression dramatically increased invasion and metastasis in gastric cancer cells. Further mechanistic research showed that hTERT directly regulates PLEKHA7 expression by binding p50 and recruiting the hTERT/p50 complex to the PLEKHA7 promoter. Increased hTERT dramatically decreased PLEKHA7 expression and promoted invasion and metastasis in gastric cancer cells. The hTERT-mediated invasion/metastasis properties at least partially depended on PLEKHA7. Our work uncovers a novel molecular mechanism underlying invasion/metastasis in gastric cancer orchestrated by hTERT and p50.

In Vivo Anchoring Bis-Pyrene Probe for Molecular Imaging of Early Gastric Cancer by Endoscopic Techniques.

With the development of blue laser endoscopy (BLE) technique, it's often used to diagnose early gastric cancer (EGC) by the morphological changes of blood vessels through BLE. However, EGC is still not obvious to identify, resulting in a high rate of missed diagnosis. Molecular imaging can show the changes in early tumors at molecular level, which provides a possibility for diagnosing EGC. Therefore, developing a probe that visually monitors blood vessels of EGC under BLE is particularly necessary. Herein, a bis-pyrene (BP) based nanoprobe (BP-FFVLK-(PEG)-RGD, M1 ) is designed, which can target angiogenesis and self-assemble into fibers in situ, resulting in stable and long-term retention in tumor. Moreover, M1 probe can emit yellow-green fluorescence for imaging under BLE. M1 probe is confirmed to steadily remain in tumor for up to 96 hours in mice transplanted subcutaneously. In addition, the M1 probe is able to target angiogenesis for molecular imaging of isolated human gastric cancer tissue under BLE. Finally, M1 probe i.v. injected into primary gastric cancer model rabbits successfully highlighted the tumor site under BLE, which is confirmed by pathological analysis. It's the first time to develop a probe for diagnosing EGC by visualizing angiogenesis under BLE, showing great clinical significance.

Identification and Characterization of Circular RNAs Involved in the Flower Development and Senescence of Rhododendron delavayi Franch

Floral development and senescence are a crucial determinant for economic and ornamental value. CircRNAs play an essential role in regulating plant growth and development; however, there is no systematic identification of circRNAs during the lifespan of flowers. This study aims to explore the expression profile and functional role of circRNAs in the full flowering stages of Rhododendron delavayi Franch. We carried out transcriptome sequencing of the six stages of Rhododendron delavayi Franch flowers to identify the circular RNA expression profile. In addition, using bioinformatics methods, we explored the functions of circRNAs, including analysis of the circRNA-miRNA-mRNA network, short time-series expression miner (STEM), and so on. We identified 146 circRNAs, of which 79 were differentially expressed from the budding to fading stages. Furthermore, using STEM analysis, one of the 42 circRNA expression model profiles was significantly upregulated during the senescence stage, including 16 circRNAs. Additionally, 7 circRNA-miRNA-mRNA networks were constructed with 10 differentially expressed circRNAs, in which some target mRNA may regulate the development and senescence of the Rhododendron flowers. Finally, by analyzing the correlation between circRNAs and mRNA, combined with existing reports, we proposed that circRNAs play a regulatory role during flower development and senescence by mediating the jasmonate signaling pathway. Overall, these results provide new clues to the potential mechanism of circRNAs acting as novel post-transcriptional regulators in the development and senescence process of flowers.

Development and Validation of an Artificial Intelligence Model for Small Bowel Capsule Endoscopy Video Review.

Importance: Reading small bowel capsule endoscopy (SBCE) videos is a tedious task for clinicians, and a new method should be applied to solve the situation. Objectives: To develop and evaluate the performance of a convolutional neural network algorithm for SBCE video review in real-life clinical care. Design, Setting, and Participants: In this multicenter, retrospective diagnostic study, a deep learning neural network (SmartScan) was trained and validated for the SBCE video review. A total of 2927 SBCE examinations from 29 medical centers were used to train SmartScan to detect 17 types of CE structured terminology (CEST) findings from January 1, 2019, to June 30, 2020. SmartScan was later validated with conventional reading (CR) and SmartScan-assisted reading (SSAR) in 2898 SBCE examinations collected from 22 medical centers. Data analysis was performed from January 25 to December 31, 2021. Exposure: An artificial intelligence-based tool for interpreting clinical images of SBCE. Main Outcomes and Measures: The detection rate and efficiency of CEST findings detected by SSAR and CR were compared. Results: A total of 5825 SBCE examinations were retrospectively collected; 2898 examinations (1765 male participants [60.9%]; mean [SD] age, 49.8 [15.5] years) were included in the validation phase. From a total of 6084 CEST-classified SB findings, SSAR detected 5834 findings (95.9%; 95% CI, 95.4%-96.4%), significantly higher than CR, which detected 4630 findings (76.1%; 95% CI, 75.0%-77.2%). SmartScan-assisted reading achieved a higher per-patient detection rate (79.3% [2298 of 2898]) for CEST findings compared with CR (70.7% [2048 of 2298]; 95% CI, 69.0%-72.3%). With SSAR, the mean (SD) number of images (per SBCE video) requiring review was reduced to 779.2 (337.2) compared with 27 910.8 (12 882.9) with CR, for a mean (SD) reduction rate of 96.1% (4.3%). The mean (SD) reading time with SSAR was shortened to 5.4 (1.5) minutes compared with CR (51.4 [11.6] minutes), for a mean (SD) reduction rate of 89.3% (3.1%). Conclusions and Relevance: This study suggests that a convolutional neural network-based algorithm is associated with an increased detection rate of SBCE findings and reduced SBCE video reading time.

Role of lncSLCO1C1 in gastric cancer progression and resistance to oxaliplatin therapy.

BACKGROUND: Gastric carcinoma (GC) is one of the most deadly diseases due to tumour metastasis and resistance to therapy. Understanding the molecular mechanism of tumour progression and drug resistance will improve therapeutic efficacy and develop novel intervention strategies. METHODS: Differentially expressed long non-coding RNAs (lncRNAs) in clinical specimens were identified by LncRNA microarrays and validated in different clinical cohorts by quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation and bioinformatics analysis. Biological functions of lncRNA were investigated by using cell proliferation assays, migration assays, xenograft tumour models and bioinformatics analysis. Effects of lncSLCO1C1 on GC cell survival were assessed by comet assays and immunofluorescence assays. Underlying molecular mechanisms were further explored by using a number of technologies including RNA pull-down, mass spectrometry analysis, RNA immunoprecipitation, co-immunoprecipitation, miRNA sequencing, luciferase reporter assays and molecular modelling. RESULTS: LncSLCO1C1 was highly upregulated in GC tissue samples and associated with GC patients' poor overall survival. Overexpression of lncSLCO1C1 promoted proliferation and migration, whereas decreased lncSLCO1C1 expression produced the opposite effects. lncSLCO1C1 also mediated tumour resistance to chemotherapy with oxaliplatin by reducing DNA damage and increasing cell proliferation. Despite sequence overlapping between lncSLCO1C1 and PDE3A, alternations of PDE3A expression had no effect on the GC cell progression, indicating that lncSLCO1C1, not PDE3A, related with the progression of GC cells. Mechanistically, lncSLCO1C1 serves as a scaffold for the structure-specific recognition protein 1 (SSRP1)/H2A/H2B complex and regulates the function of SSRP1 in reducing DNA damage. Meanwhile, lncSLCO1C1 functions as a sponge to adsorb miR-204-5p and miR-211-5p that target SSRP1 mRNA, and thus increases SSRP1 expression. Patients with high expressions of both lncSLCO1C1 and SSRP1 have poor overall survival, highlighting the role of lncSLCO1C1 in GC progression. CONCLUSIONS: LncSLCO1C1 promotes GC progression by enhancing cell growth and preventing DNA damage via interacting and scaffolding the SSRP1/H2A/H2b complex and absorbing both miR-211-5p and miR-204-5p to increase SSRP1 expression.

Gut Microbiota Associated With Effectiveness And Responsiveness to Mindfulness-Based Cognitive Therapy in Improving Trait Anxiety.

Objective: Mindfulness-based interventions have been widely demonstrated to be effective in reducing stress, alleviating mood disorders, and improving quality of life; however, the underlying mechanisms remained to be fully understood. Along with the advanced research in the microbiota-gut-brain axis, this study aimed to explore the impact of gut microbiota on the effectiveness and responsiveness to mindfulness-based cognitive therapy (MBCT) among high trait anxiety populations. Design: A standard MBCT was performed among 21 young adults with high trait anxiety. A total of 29 healthy controls were matched for age and sex. The differences in gut microbiota between the two groups were compared. The changes in fecal microbiota and psychological indicators were also investigated before and after the intervention. Results: Compared with healthy controls, we found markedly decreased bacterial diversity and distinctive clusters among high trait anxiety populations, with significant overgrowth of bacteria such as Streptococcus, Blautia, and Romboutsia, and a decrease in genera such as Faecalibacterium, Coprococcus_3, and Lachnoclostridium. Moreover, MBCT attenuated trait anxiety and depression, improved mindfulness and resilience, and increased the similarity of gut microbiota to that of healthy controls. Notably, a high presence of intestinal Subdoligranulum pre-MBCT was associated with increased responsiveness to MBCT. Decreases in Subdoligranulum post-MBCT were indicative of ameliorated trait anxiety. The tryptophan metabolism pathways were significantly over-represented among high responders compared to low responders. Conclusion: The significantly increased diversity post-MBCT added evidence to gut-brain communication and highlighted the utility of mycobiota-focused strategies for promoting the effectiveness and responsiveness of the MBCT to improve trait anxiety. Clinical Trial Registration: chictr.org.cn, ChiCTR1900028389.