Saturation mutagenesis-reinforced functional assays for disease-related genes.

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods are obstacles for achieving genome-wide resolution of variants in disease-related genes. Our framework, saturation mutagenesis-reinforced functional assays (SMuRF), offers simple and cost-effective saturation mutagenesis paired with streamlined functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for all possible coding single-nucleotide variants, which aid in resolving clinically reported variants of uncertain significance. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Overall, our approach enables variant-to-function insights for disease genes in a cost-effective manner that can be broadly implemented by standard research laboratories.

Quantifying constraint in the human mitochondrial genome.

Mitochondrial DNA (mtDNA) has an important yet often overlooked role in health and disease. Constraint models quantify the removal of deleterious variation from the population by selection and represent powerful tools for identifying genetic variation that underlies human phenotypes1-4. However, nuclear constraint models are not applicable to mtDNA, owing to its distinct features. Here we describe the development of a mitochondrial genome constraint model and its application to the Genome Aggregation Database (gnomAD), a large-scale population dataset that reports mtDNA variation across 56,434 human participants5. Specifically, we analyse constraint by comparing the observed variation in gnomAD to that expected under neutrality, which was calculated using a mtDNA mutational model and observed maximum heteroplasmy-level data. Our results highlight strong depletion of expected variation, which suggests that many deleterious mtDNA variants remain undetected. To aid their discovery, we compute constraint metrics for every mitochondrial protein, tRNA and rRNA gene, which revealed a range of intolerance to variation. We further characterize the most constrained regions within genes through regional constraint and identify the most constrained sites within the entire mitochondrial genome through local constraint, which showed enrichment of pathogenic variation. Constraint also clustered in three-dimensional structures, which provided insight into functionally important domains and their disease relevance. Notably, we identify constraint at often overlooked sites, including in rRNA and noncoding regions. Last, we demonstrate that these metrics can improve the discovery of deleterious variation that underlies rare and common phenotypes.

Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

Preparing the key metabolite of Z-ligustilide in vivo by a specific electrochemical reaction.

The key in vivo metabolites of a drug play an important role in its efficacy and toxicity. However, due to the low content and instability of these metabolites, they are hard to obtain through in vivo methods. Electrochemical reactions can be an efficient alternative to biotransformation in vivo for the preparation of metabolites. Accordingly, in this study, the metabolism of Z-ligustilide was investigated in vitro by electrochemistry coupled online to mass spectrometry. This work showed that five oxidation products of the electrochemical reaction were detected and that two of the oxidation products (senkyunolide I and senkyunolide H) were identified from liver microsomal incubation as well. Furthermore, after intragastric administration of Z-ligustilide in rats, senkyunolide I and senkyunolide H were detected in the rat plasma and liver, while 6,7-epoxyligustilide, a key intermediate metabolite of Z-ligustilide, was difficult to detect in vivo. By contrast, 6,7-epoxyligustilide was obtained from the electrochemical reaction. In addition, for the first time, 6 mg of 6,7-epoxyligustilide was prepared from 120 mg of Z-ligustilide. Therefore, electrochemical reactions represent an efficient laboratory method for preparing key drug metabolites.

High-throughput assays to assess variant effects on disease.

Interpreting the wealth of rare genetic variants discovered in population-scale sequencing efforts and deciphering their associations with human health and disease present a critical challenge due to the lack of sufficient clinical case reports. One promising avenue to overcome this problem is deep mutational scanning (DMS), a method of introducing and evaluating large-scale genetic variants in model cell lines. DMS allows unbiased investigation of variants, including those that are not found in clinical reports, thus improving rare disease diagnostics. Currently, the main obstacle limiting the full potential of DMS is the availability of functional assays that are specific to disease mechanisms. Thus, we explore high-throughput functional methodologies suitable to examine broad disease mechanisms. We specifically focus on methods that do not require robotics or automation but instead use well-designed molecular tools to transform biological mechanisms into easily detectable signals, such as cell survival rate, fluorescence or drug resistance. Here, we aim to bridge the gap between disease-relevant assays and their integration into the DMS framework.

Efficient selective adsorption of Cr(VI) by S-doped porous carbon prepared from industrial lignin: Waste increment and wastewater treatment.

Industrial lignin is a waste product of the paper industry, which contains a large amount of oxygen group structure, and can be used to treat industrial wastewater containing Cr(VI). However, lignin has very low reactivity, so how to enhance its adsorption performance is a major challenge at present. In this study, a two-stage hydrothermal and activation strategy was used to activate the lignin activity and doping S element to prepare high-performance S-doped lignin-based polyporous carbon (S-LPC). The results show that the surface of S-LPC is rich in S and O groups and has a well-developed pore structure, which is very beneficial to Cr(VI) uptake -reduction and mass transfer on the material. In the wastewater, the utmost adsorption potential of Cr(VI) by S-LPC achieved 882.83 mg/g. After 7 cycles of regeneration, the adsorption of S-LPC decreased by only approximately 18 %. Ion competition experiments showed that S-LPC has excellent specificity for Cr(VI) adsorption. In factory wastewater, the adsorption performance of S-LPC for Cr(VI) remained above 95 %, which shows the excellent performance of S-LPC in practical applications. The results are of great significance for green chemical utilization of waste lignin, treatment of industrial wastewater and sustainable development.

Two-stage hydrothermal oxygenation for efficient removal of Cr(VI) by starch-based polyporous carbon: Wastewater application and removal mechanism.

Cr(VI) is of concern because of its high mobility and toxicity. In this work, a two-stage hydrothermal strategy was used to activate the O sites of starch, and by inserting K-ion into the pores, starch-based polyporous carbon (S-PC) adsorption sites was synthesized for removal of Cr(VI). Physicochemical characterization revealed that the O content of the S-PC reached 20.66 % after activation, indicating that S-PC has excellent potential for adsorption of Cr(VI). The S-PC removal rate for 100 mg/L Cr(VI) was 96.29 %, and the adsorption capacity was 883.86 mg/g. Moreover, S-PC showed excellent resistance to interference, and an equal concentration of hetero-ions reduced the activity by less than 5 %. After 8 cycles of factory wastewater treatment, the S-PC maintained 81.15 % of its original activity, which indicated the possibility of practical application. Characterization and model analyses showed that the removal of Cr(VI) from wastewater by the S-PC was due to CC, δ-OH, ν-OH, and C-O-C groups, and the synergistic effect of adsorption and reduction was the key to the performance. This study provides a good solution for treatment of Cr(VI) plant wastewater and provides a technical reference for the use of biological macromolecules such as starch in the treatment of heavy metals.

Potential mechanisms underlying podophyllotoxin-induced cardiotoxicity in male rats: toxicological evidence chain (TEC) concept.

Introduction: Podophyllotoxin (PPT) is a high-content and high-activity compound extracted from the traditional Chinese medicinal plant Dysosma versipellis (DV) which exhibits various biological activities. However, its severe toxicity limits its use. In clinical settings, patients with DV poisoning often experience adverse reactions when taking large doses in a short period. The heart is an important toxic target organ, so it is necessary to conduct 24-h acute cardiac toxicity studies on PPT to understand its underlying toxicity mechanism. Methods: Based on the concept of the toxicological evidence chain (TEC), we utilized targeted metabolomic and transcriptomic analyses to reveal the mechanism of the acute cardiotoxicity of PPT. The manifestation of toxicity in Sprague-Dawley rats, including changes in weight and behavior, served as Injury Phenotype Evidence (IPE). To determine Adverse Outcomes Evidence (AOE), the hearts of the rats were evaluated through histopathological examination and by measuring myocardial enzyme and cardiac injury markers levels. Additionally, transcriptome analysis, metabolome analysis, myocardial enzymes, and cardiac injury markers were integrated to obtain Toxic Event Evidence (TEE) using correlation analysis. Results: The experiment showed significant epistaxis, hypokinesia, and hunched posture in PPT group rats within 24 h after exposure to 120 mg/kg PPT. It is found that PPT induced cardiac injury in rats within 24 h, as evidenced by increased serum myocardial enzyme levels, elevated concentrations of cardiac injury biomarkers, and altered cardiac cell morphology, all indicating some degree of cardiac toxicity. Transcriptome analysis revealed that primary altered metabolic pathway was arachidonic acid metabolism after PPT exposure. Cyp2e1, Aldob were positively correlated with differential metabolites, while DHA showed positive correlation with differential genes Fmo2 and Timd2, as well as with heart injury markers BNP and Mb. Conclusion: This study comprehensively evaluated cardiac toxicity of PPT and initially revealed the mechanism of PPT-induced acute cardiotoxicity, which involved oxidative stress, apoptosis, inflammatory response, and energy metabolism disorder.

Lama1 upregulation prolongs the lifespan of the dyH/dyH mouse model of LAMA2-related congenital muscular dystrophy.

LAMA2-related congenital muscular dystrophy (LAMA2-CMD), characterized by laminin-α2 deficiency, is debilitating and ultimately fatal. To date, no effective therapy has been clinically available. Laminin-α1, which shares significant similarities with laminin-α2, has been proven as a viable compensatory modifier. To evaluate its clinical applicability, we establish a Lama2 exon-3-deletion mouse model (dyH/dyH). The dyH/dyH mice exhibit early lethality and typical LAMA2-CMD phenotypes, allowing the evaluation of various endpoints. In dyH/dyH mice treated with synergistic activation mediator-based CRISPRa-mediated Lama1 upregulation, a nearly doubled median survival is observed, as well as improvements in weight and grip. Significant therapeutical effects are revealed by MRI, serum biochemical indices, and muscle pathology studies. Treating LAMA2-CMD with LAMA1 upregulation is feasible, and early intervention can alleviate symptoms and extend lifespan. Additionally, we reveal the limitations of LAMA1 upregulation, including high-dose mortality and non-sustained expression, which require further optimization in future studies.

Comparative genomics of macaques and integrated insights into genetic variation and population history.

The crab-eating macaques ( Macaca fascicularis ) and rhesus macaques ( M. mulatta ) are widely studied nonhuman primates in biomedical and evolutionary research. Despite their significance, the current understanding of the complex genomic structure in macaques and the differences between species requires substantial improvement. Here, we present a complete genome assembly of a crab-eating macaque and 20 haplotype-resolved macaque assemblies to investigate the complex regions and major genomic differences between species. Segmental duplication in macaques is ∼42% lower, while centromeres are ∼3.7 times longer than those in humans. The characterization of ∼2 Mbp fixed genetic variants and ∼240 Mbp complex loci highlights potential associations with metabolic differences between the two macaque species (e.g., CYP2C76 and EHBP1L1 ). Additionally, hundreds of alternative splicing differences show post-transcriptional regulation divergence between these two species (e.g., PNPO ). We also characterize 91 large-scale genomic differences between macaques and humans at a single-base-pair resolution and highlight their impact on gene regulation in primate evolution (e.g., FOLH1 and PIEZO2 ). Finally, population genetics recapitulates macaque speciation and selective sweeps, highlighting potential genetic basis of reproduction and tail phenotype differences (e.g., STAB1 , SEMA3F , and HOXD13 ). In summary, the integrated analysis of genetic variation and population genetics in macaques greatly enhances our comprehension of lineage-specific phenotypes, adaptation, and primate evolution, thereby improving their biomedical applications in human diseases.

The effects of physiotherapeutic scoliosis-specific exercise on idiopathic scoliosis in children and adolescents: a systematic review and meta-analysis.

BACKGROUND: Physiotherapeutic scoliosis-specific exercise (PSSE) is recommended by SOSORT as the first step in the treatment of adolescent idiopathic scoliosis (AIS). However, a thorough summary and meta-analysis of the evidence for the effectiveness of PSSE is lacking. OBJECTIVE: To summarise the up-to-date evidence on the efficacy of PSSE in AIS compared with the other non-surgical therapies. DATA SOURCES: The PubMed, Web of Science, Cochrane, Scopus, Embase and CNKI databases were systematically searched from 1 January 2012 to 1 November 2022. STUDY SELECTION: Controlled trials comparing the effects of PSSE and other non-surgical therapies on improving Cobb angle and quality of life in young people aged 6-18 years were included. DATA SYNTHESIS: Three researchers independently extracted data and evaluated methodological quality. Meta-analysis was performed where possible; otherwise, descriptive syntheses were reported. RESULTS: Seventeen studies with a total of 930 participants (76% female) were included. Among them, ten studies were RCTs. Six studies were of excellent quality. Thirteen studies were included in the meta-analysis. PSSE corrected the Cobb angle in patients better than other non-surgical therapies (I2 = 82%, MD = -2.82, 95%CI = -4.17 to -1.48, P < 0.01). PSSE was more effective than brace in improving patients' pain, self-image and mental health. Patients with a Risser grade of 0-3 or who had never received brace therapy had better outcomes. CONCLUSION: Evidence from higher quality studies suggests that PSSE was superior to general exercise and conventional therapy for correcting the Cobb angle in AIS. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO ID CRD42022345157 CONTRIBUTION OF THE PAPER.

Study on the Process Characteristics Based on Joule Heat of Sliding-Pressure Additive Manufacturing.

This study developed an experimental system based on Joule heat of sliding-pressure additive manufacturing (SP-JHAM), and Joule heat was used for the first time to accomplish high-quality single-layer printing. The roller wire substrate is short-circuited, and Joule heat is generated to melt the wire when the current passes through. Through the self-lapping experimental platform, single-factor experiments were designed to study the effects of power supply current, electrode pressure, contact length on the surface morphology and cross-section geometric characteristics of the single-pass printing layer. Through the Taguchi method, the effect of various factors was analyzed, the optimal process parameters were obtained, and the quality was detected. The results show that with the current increase, the aspect ratio and dilution rate of a printing layer increase within a given range of process parameters. In addition, with the increase in pressure and contact length, the aspect ratio and dilution ratio decrease. Pressure has the greatest effect on the aspect ratio and dilution ratio, followed by current and contact length. When a current of 260 A, a pressure of 0.60 N and a contact length of 1.3 mm are applied, a single track with a good appearance, whose surface roughness Ra is 3.896 µm, can be printed. Additionally, the wire and the substrate are completely metallurgically bonded with this condition. There are also no defects such as air holes and cracks. This study verified the feasibility of SP-JHAM as a new additive manufacturing strategy with high quality and low cost, and provided a reference for developing additive manufacturing technology based on Joule heat.

Enhancement of Cr(VI) adsorption on lignin-based carbon materials by a two-step hydrothermal strategy: Performance and mechanism.

Cr(VI) is a carcinogenic heavy metal that forms an oxygen-containing anion, which is difficult to remove from water by adsorbents. Here, industrial alkali lignin was transformed into a Cr(VI) adsorbent (N-LC) by using a two-step hydrothermal strategy. The characterization results of the adsorbent showed that O and N were uniformly distributed on the surface of the adsorbent, resulting in a favorable morphology and structure. The Cr(VI) adsorption of N-LC was 13.50 times that of alkali lignin, and the maximum was 326.10 mg g-1, which confirmed the superiority of the two-step hydrothermal strategy. After 7 cycles, the adsorption of N-LC stabilized at approximately 62.18 %. In addition, in the presence of coexisting ions, N-LC showed a selective adsorption efficiency of 85.47 % for Cr(VI), which is sufficient to support its application to actual wastewaters. Model calculations and characterization showed that N and O groups were the main active factors in N-LC, and CO, -OH and pyridinic-N were the main active sites. This study provides a simple and efficient method for the treatment of heavy metals and the utilization of waste lignin, which is expected to be widely applied in the environmental, energy and chemical industries.

Trinuclear Fe Clusters for Highly Efficient CO2 Photoreduction.

Photocatalytic reduction of CO2 into valuable chemicals or fuels is considered a promising solution to mitigate the energy crisis. In this work, efficient CO2 to CO conversion was achieved, accompanied by a class of trinuclear Fe clusters as photocatalysts. Under optimal conditions, the highest catalytic rate could be up to 140.9 µmol/h in 6 h with the assistance of photosensitizers (PS). The trinuclear Fe Clusters can be used as secondary building units to construct Fe-based metal-organic frameworks (MOFs). However, the catalytic activity of Fe-based MOFs is weaker than that of clusters in both the cases of extra PS-assisted MOFs and integrated PS into MOFs. The simpler synthesis, lower cost, and higher catalytic activity make the Fe clusters a better catalyst. Additionally, steady-state fluorescence tests confirmed the transfer of photogenerated electrons from PS to the clusters during the photocatalytic reaction.

CLMP is a tumor suppressor that determines all-trans retinoic acid response in colorectal cancer.

CAR-like membrane protein (CLMP) is a tight junction-associated protein whose mutation is associated with congenital short bowel syndrome (CSBS), but its functions in colorectal cancer (CRC) remain unknown. Here, we demonstrate that CLMP is rarely mutated but significantly decreased in CRC patients, and its deficiency accelerates CRC tumorigenesis, growth, and resistance to all-trans retinoic acid (ATRA). Mechanistically, CLMP recruits ß-catenin to cell membrane, independent of cadherin proteins. CLMP-mediated ß-catenin translocation inactivates Wnt(Wingless and INT-1)/ß-catenin signaling, thereby suppressing CRC tumorigenesis and growth in ApcMin/+, azoxymethane/dextran sodium sulfate (AOM/DSS), and orthotopic CRC mouse models. As a direct target of Wnt/ß-catenin, cytochrome P450 hydroxylase A1 (CYP26A1)-an enzyme that degrades ATRA to a less bioactive retinoid-is upregulated by CLMP deficiency, resulting in ATRA-resistant CRC that can be reversed by administering CYP26A1 inhibitor. Collectively, our data identify the anti-CRC role of CLMP and suggest that CYP26A1 inhibitor enable to boost ATRA's therapeutic efficiency.

Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods hamper crowd-sourcing approaches toward genome-wide resolution of variants in disease-related genes. Our framework, Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these issues by offering simple and cost-effective saturation mutagenesis, as well as streamlining functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for over 99.8% of all possible coding single nucleotide variants and resolved 310 clinically reported variants of uncertain significance with high confidence, enhancing clinical variant interpretation in dystroglycanopathies. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Our approach opens new directions for enabling variant-to-function insights for disease genes in a manner that is broadly useful for crowd-sourcing implementation across standard research laboratories.

Preschool Children's Physical Activity and Community Environment: A Cross-Sectional Study of Two Cities in China.

Research on the relationship between preschool children's physical activity (PA) and community environment is limited and inconclusive, yet understanding this relationship is important to acquire sufficient information to guide the development of intervention programs. This study aims to objectively measure preschool children's PA and examine associations between PA and the community environments. A total of 471 preschool children aged 3-6 years old were recruited from eight kindergartens in Beijing and Zhengzhou. PA was measured by accelerometers. Light PA (LPA), moderate PA (MPA), and vigorous PA (VPA) were computed on the basis of cutoff points developed for preschool children. Moderate-to-vigorous PA (MVPA), and total PA (TPA) were obtained by calculation. Children's active transportation modes were indicated by the frequency of active trips (FAT) reported by parents. The community environment was collected by parental scales. Multivariate linear regression was used to analyze the associations between PA and the community environment. In total, 304 preschool children (mean age 5.07 ± 0.94 years, 50.66% boys) were included in the final analysis. Children spent an average of 77.58 ± 18.78 min/day in MVPA and 173.26 ± 30.38 min/day in TPA. Linear regression showed that 'parental perception of appropriate walking distance' was associated with nearly half of the indicators of the children's PA. 'Public activity facilities near the community' was associated with FAT for overall children (B = 0.099, 95% CI = 0.014, 0.183). 'Community transportation environment' was associated with overall children's average day LPA (B = 4.034, 95% CI = 0.012, 8.056), weekend LPA (B = 8.278, 95% CI = 1.900, 14.657), MPA (B = 4.485, 95% CI = 0.613, 8.357), TPA (B = 14.777, 95% CI = 2.130, 27.424), and FAT for girls (B = -0.223, 95% CI = -0.443, -0.003). Furthermore, 'community personal safety' was associated with boys' weekday VPA (B = -3.012, 95% CI = -5.946, 0.079). Parental perception of appropriate walking distances, improvement of PA facilities, community personal safety, and the community transportation environment all contribute to the prevention of preschool children's PA patterns deterioration.

Nuclease-Deficient Clustered Regularly Interspaced Short Palindromic Repeat-Based Approaches for In Vitro and In Vivo Gene Activation.

Clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been adapted to achieve a wide range of genome modifications, including transcription regulation. The focus of this review is on the application of CRISPR-based platforms such as nuclease-deficient Cas9 and Cas12a, to achieve targeted gene activation. We review studies to date that have used CRISPR-based activation technology for the elucidation of biological mechanism and disease correction, as well as its application in genetic screens as a powerful tool for high-throughput genotype-phenotype mapping. In addition to our synthesis and critical analysis of published studies, we explore key considerations for the potential clinical translation of CRISPR-based activation technology.

Barley C-Hordein as the Calibrant for Wheat Gluten Quantification.

The lack of certified reference materials has been one major challenge for gluten quantification in gluten-free products. In this study, the feasibility of using barley C-hordein as the calibrant for wheat gluten in R5 sandwich enzyme-linked immunosorbent assay (ELISA) was investigated. The gluten composition and total gluten R5 reactivity ranged largely depending on the genotypes and the growing environment. The conversion factor of gliadin to gluten averaged 1.31 for common wheat, which is smaller than the theoretical factor of 2. Each gluten group had varying reactivity against the R5 antibody, where ω1.2-, γ- and α-gliadins were the main reactive groups from wheat gluten. A mixture of wheat cultivars or one single cultivar as the reference material can be difficult to keep current. Based on the average R5 reactivity of total gluten from the 27 common wheat cultivars, here we proposed 10% C-hordein mixed with an inert protein as the calibrant for wheat gluten quantification. In spiking tests of gluten-free oat flour and biscuits, calibration using 10% C-hordein achieved the same recovery as the gliadin standard with its cultivar-specific conversion factor. For its good solubility and good affinity to the R5 antibody, the application of C-hordein increases the probability of developing a series of reference materials for various food matrices.

Study on the mechanisms of "Xiaoerhuashi Pill, XP" in intervening the health status of high-calorie diet animals.

ETHNOPHARMACOLOGICAL RELEVANCE: "Xiaoerhuashi Pill, XP", with a history of 30 years in China, was included in the first part of the 2015 edition of the Chinese Pharmacopoeia and is widely used in the treatment for pediatric diseases in clinical application. Its main indications include the accumulation of heat caused by food stagnation in children, which has the effect of digestive stagnation and purge heat to relax the bowels. AIM OF THE STUDY: High-calorie diet, closely related to the occurrence and development of multiple diseases, is an unhealthy status of life. However, there is no effective intervention in clinic. Thus, based on animal experiments and bioinformatics, this study aims to explore the potential mechanisms of action of Chinese patent medicine- "Xiaoerhuashi Pill, XP" in the intervention of high-calorie diet. MATERIALS AND METHODS: A high-calorie diet model was prepared by 3-week-old rats. The defecation and intestinal mucosal morphology were observed after intragastric administration of "Xiaoerhuashi Pill, XP". The components of "Xiaoerhuashi Pill, XP" were obtained by chromatography-mass spectrometry, with the corresponding targets obtained by database and target fishing. The key effects substances were obtained by molecular docking, with the obtaining of the ore pathway of "Xiaoerhuashi Pill, XP" in intervention of high-calorie diet based on the enrichment analysis. RESULTS: "Xiaoerhuashi Pill, XP" can actively interfere with defecation and intestinal mucosal structures in high-calorie diet animals. A total of 37 substances were identified in the pediatric digestion solution, and 356 target proteins were mapped, 25 of which were associated with a high-calorie diet. Overall, the analysis shows that the highest degree of integration was quercetin and PON1 protein, with the highest enrichment of insulin resistance pathway. CONCLUSION: "Xiaoerhuashi Pill, XP" can intervene in the health status of high-calorie diet animals. Integration of quercetin and PON1 protein can regulate lipid levels, which may be the key mechanisms of action in "Xiaoerhuashi Pill, XP". The mechanisms, more specifically, may be related to the regulation of pancreas islet function, thus providing a reference for the clinical application of "Xiaoerhuashi Pill, XP", clinical intervention of high-calorie diet and new drug development.