OBJECTIVE To investigate the effect and mechanism of Jingangteng capsules in the treatment of non-alcoholic fatty liver disease （NAFLD）. METHODS Thirty-two SD rats were randomly divided into normal group and modeling group. The modeling group was fed a high-fat diet to establish a NAFLD model. The successfully modeled rats were then randomly divided into model group， atorvastatin group[positive control， 2 mg/（kg·d）]， and Jingangteng capsules low- and high-dose groups [0.63 and 2.52 mg/（kg·d）]， with 6 rats in each group. The pathological changes of the liver were observed by hematoxylin-eosin staining and oil red O staining. Enzyme-linked immunosorbent assay was performed to determine the serum levels of triglycerides （TG）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， alanine transaminase （ALT）， aspartate transaminase （AST）， tumour necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， IL-6， IL-18. 16S rDNA amplicon sequencing and metabolomics techniques were applied to explore the effects of Jingangteng capsules on gut microbiota and metabolisms in NAFLD rats. Based on the E-mail：591146765@qq.com metabolomics results， Western blot analysis was performed to detect proteins related to the nuclear factor kappa-B （NF-κB）/NOD-like receptor family protein 3 （NLRP3） signaling pathway in the livers of NAFLD rats. RESULTS The experimental results showed that Jingangteng capsules could significantly reduce the serum levels of TG， TC， LDL-C， AST， ALT， TNF-α， IL-1β， IL-6， IL-18， while increased the level of HDL-C， and alleviated the hepatic cellular steatosis and inflammatory infiltration in NAFLD rats. They could regulate the gut microbiota disorders in NAFLD rats， significantly increased the relative abundance of Romboutsia and Oscillospira， and significantly decreased the relative abundance of Blautia （P＜0.05）. They also regulated metabolic disorders primarily by affecting secondary bile acid biosynthesis， fatty acid degradation， O-antigen nucleotide sugar biosynthesis， etc. Results of Western blot assay showed that they significantly reduced the phosphorylation levels of NF-κB p65 and NF-κB inhibitor α， and the protein expression levels of NLRP3， caspase-1 and ASC （P＜0.05 or P＜0.01）. CONCLUSIONS Jingangteng capsules could improve inflammation， lipid accumulation and liver injury in NAFLD rats， regulate the disorders of gut microbiota and metabolisms， and inhibit NF-κB/NLRP3 signaling pathway. Their therapeutic effects against NAFLD are mediated through the inhibition of the NF-κB/NLRP3 signaling pathway.

Objective:To establish a TaqMan-MGB probe-based method for detecting the polymorphic loci C677T and A1298C of the MTHFR gene.Methods:Specific primers and TaqMan-MGB probes targeting the C677T and A1298C polymorphic loci of the MTHFR gene were designed and optimized based on the gene sequence information.A real-time quantitative PCR detection system was established.Gradient dilution experiments were conducted to determine the limit of detection,and reproducibility experiments were performed to evaluate detection consistency.Specificity was validated using wild-type and mutant plasmid templates.The method was applied to detect 56 clinical samples,and its accuracy and practicality were assessed through comparison with traditional Sanger sequencing.Results:The TaqMan-MGB probe method demonstrated high specificity for detecting the C677T and A1298C loci,with no cross-reactivity between wild-type and mutant probes,enabling accurate genotype differentiation.Sensitivity experiments revealed detection limits of 1.13 × 103 copies/μL for C677T and 8.39 × 101 copies/μL for A1298C.Reproducibility experiments showed coefficients of variation below 1％,indicating stable and reliable results.Among the 56 clinical samples,the overall detection rate for the C677T locus was 86.99％,and for the A1298C locus,it was 97.92％.The TaqMan-MGB method exhibited good concordance with Sanger sequencing results.Conclusion:The TaqMan-MGB method exhibits high specificity,sensitivity,and excellent reproducibility in detecting the polymorphic loci C677T and A1298C of the MTHFR gene,making it suitable for rapid detection in large-scale clinical samples.This method provides an effective molecular diagnostic tool for the early diagnosis and prevention of folate-related diseases.

Objective To verify the applicability of a pig STR multiplex amplification system for detecting processed foods containing pork and their digestive samples,and to evaluate its potential in food safety and forensic biological evidence analysis.Methods DNA profiles were obtained using the pig STR amplification system from food samples with different levels of processing(raw pork,boiled pork,fried pork,and sausage)and from digestive samples(rat gastric contents).The influence of processing methods on DNA integrity was assessed.The uniformity of large-scale processed ham products,the consistency of DNA profiles from different parts of the same sample,and the DNA degradation patterns after rat digestion were examined.Results STR profiling of pig DNA was successful in all tested samples.Short fragments showed high amplification stability,while long fragment signals weakened with increasing processing complexity.In processed ham products,DNA profiles were consistent across all sampled parts,with fragment drift within±0.5 bp.Analysis of rat gastric contents showed slight DNA degradation within 2 hours;after 3 hours,long fragment signals weakened,and after 4 hours,some loci signals were lost.Conclusion The pig STR multiplex amplification system exhibits excellent performance in detecting processed pork products and their digestive samples.It can meet the requirements of food traceability and forensic biological evidence analysis for processed pork,providing new insights for the advancement of forensic testing techniques in this field.

Aim To investigate the neuroprotective effect of Takeda G protein-coupled receptor-5(TGR5)activated by INT-777 on hypoxic-ischemic encephalop-athy(HIE)in neonatal rats.Methods Seven-day-old SD rats were randomly divided into the sham opera-tion group(Sham,S),model group(HIE,G),INT-777 low-dose(L),medium-dose(M),and high-dose(H)groups.The modified Rice-Vanucci method was used to construct the HIE model and Intranasal admin-istration 1 h after modeling.Short-term neurobehavioral tests were performed 48 h after modeling to evaluate the neurological function of neonatal rats,TTC staining was used to determine the volume of cerebral infarction,dry and wet specific gravity was used to determine the brain water content,ferrous ion kit was used to deter-mine the brain ferrous ion content,HE staining was used to observe the pathological damage of brain tis-sue,Nissl staining was used to observe the loss of Nissl substance,Transmission electron microscopy(TEM)was used to observe the mitochondrial morphological changes of cortical neurons,and Western blot was em-ployed to detect the expression of ferroptosis-related proteins TFR1 and GPX4.Results Compared with group S,group G had increased short-term neurobehav-ioral test consumption time,higher scores,increased cerebral infarct volume,brain water content,and brain ferrous iron content,significant brain tissue damage on the affected side,severe loss of Nissl substance,smaller neuronal mitochondria,decreased mitochondrial cris-tae,and increased expression of TFR1 and reduced ex-pression of GPX4.Compared with group G,the INT-777 administration group had a shorter consumption time for short-term neurobehavioral tests,lower scores,the cerebral infarction volume,brain water content,and brain ferrous ion content decreased,the brain tissue damage on the affected side was reduced,and there was insignificant loss of Nissl substance,larger neuronal mi-tochondrial volume,increased mitochondrial cristae,re-duced expression of TFR1,and increased expression of GPX4.Conclusions INT-777 agonist TGR5 has a protective effect against hypoxic-ischemic encephalopa-thy in neonatal rats,and its mechanism of action may be related to the inhibition of neuronal ferroptosis.

Objective:To develop national standard materials for Streptococcus pneumoniae serotypes 12F,14,17F and 22F polysaccharides.Methods:According to the method recommended by the 2020 Chinese Pharmacopoeia,the relevant limit quality control of four different serotypes of polysaccharide candidate standards was conducted.The uniformity of subpackage and stability of the candidate standard products under different conditions were ana-lyzed.Furthermore,three laboratories were organized for collaborative calibration.Results:The results showed that the quality of serotypes 12F,14,17F and 22F polysaccharides candidate standards met the requirements of the quality standard of polysaccharide in the 2020 edition of the Chinese Pharmacopoeia.The RSD between polysaccha-ride content of different candidate standard samples were less than 5.0％,indicating that the uniformity of the poly-saccharide candidate standards was better.The results from stability analysis confirmed that the polysaccharide con-centration of serotypes 12F,14,17F and 22F polysaccharide candidate standard did not change significantly after being placed at 2-8 ℃,25 and 37℃for different times and after repeated freezing and thawing three times.Compared with the test results of each candidate standard product stored at-70 ℃ for a long time,the RSD were all within 5％.The collaborative results confirmed that the RSD of the polysaccharide content in different laborato-ries were less than 12％,of which assigned the concentration of four different serotype polysaccharide candidate standards.Conclusion:Serotypes 12F,14,17F and 22F polysaccharide standards have been developed,of which can be used for quality control of pneumococcal polysaccharide vaccines in the future.

Regarding the challenge of early identification of critically ill patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD),a radiomics-clinical fusion model is proposed based on non-contrast CT images of accessory respiratory muscles to predict life-threatening conditions.A retrospective study is conducted involving 233 AECOPD patients(153 non-life-threatening and 80 life-threatening cases).Patients are divided into a training set(n=186)and a test set(n=47)at a 4:1 ratio.A total of 1 874 radiomic features are extracted from the erector spinae and pectoralis muscle regions delineated by radiologists on non-contrast CT images,and the features selection is performed using maximum relevance minimum redundancy and least absolute shrinkage and selection operator(LASSO)algorithms.Meanwhile,clinical data are analyzed with t-test and LASSO for variable screening.The selected features are input into C-support vector classification,Logistic regression,random forest,adaptive boosting(AdaBoost),and extreme gradient boosting(XGBoost)to construct radiomics model,clinical model,and fusion model.Predictive performance and clinical practicality are evaluated in the test set using receiver operating characteristic curve,area under the curve(AUC),and decision curve analysis.The radiomics-clinical fusion model built with XGBoost outperformed standalone radiomics and clinical models,achieving an AUC of 0.902(95％CI 0.846,0.994),with accuracy,sensitivity,specificity,and precision of 0.837,0.933,0.786,and 0.7,respectively.Results demonstrate that the fusion model based on the non-contrast CT radiomics of accessory respiratory muscles and clinical data exhibits promising diagnostic performance,highlighting its potential clinical significance for stratified management and preemptive critical care intervention in AECOPD patients.

Regarding the challenge of early identification of critically ill patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD),a radiomics-clinical fusion model is proposed based on non-contrast CT images of accessory respiratory muscles to predict life-threatening conditions.A retrospective study is conducted involving 233 AECOPD patients(153 non-life-threatening and 80 life-threatening cases).Patients are divided into a training set(n=186)and a test set(n=47)at a 4:1 ratio.A total of 1 874 radiomic features are extracted from the erector spinae and pectoralis muscle regions delineated by radiologists on non-contrast CT images,and the features selection is performed using maximum relevance minimum redundancy and least absolute shrinkage and selection operator(LASSO)algorithms.Meanwhile,clinical data are analyzed with t-test and LASSO for variable screening.The selected features are input into C-support vector classification,Logistic regression,random forest,adaptive boosting(AdaBoost),and extreme gradient boosting(XGBoost)to construct radiomics model,clinical model,and fusion model.Predictive performance and clinical practicality are evaluated in the test set using receiver operating characteristic curve,area under the curve(AUC),and decision curve analysis.The radiomics-clinical fusion model built with XGBoost outperformed standalone radiomics and clinical models,achieving an AUC of 0.902(95％CI 0.846,0.994),with accuracy,sensitivity,specificity,and precision of 0.837,0.933,0.786,and 0.7,respectively.Results demonstrate that the fusion model based on the non-contrast CT radiomics of accessory respiratory muscles and clinical data exhibits promising diagnostic performance,highlighting its potential clinical significance for stratified management and preemptive critical care intervention in AECOPD patients.

Objective:To develop national standard materials for Streptococcus pneumoniae serotypes 12F,14,17F and 22F polysaccharides.Methods:According to the method recommended by the 2020 Chinese Pharmacopoeia,the relevant limit quality control of four different serotypes of polysaccharide candidate standards was conducted.The uniformity of subpackage and stability of the candidate standard products under different conditions were ana-lyzed.Furthermore,three laboratories were organized for collaborative calibration.Results:The results showed that the quality of serotypes 12F,14,17F and 22F polysaccharides candidate standards met the requirements of the quality standard of polysaccharide in the 2020 edition of the Chinese Pharmacopoeia.The RSD between polysaccha-ride content of different candidate standard samples were less than 5.0％,indicating that the uniformity of the poly-saccharide candidate standards was better.The results from stability analysis confirmed that the polysaccharide con-centration of serotypes 12F,14,17F and 22F polysaccharide candidate standard did not change significantly after being placed at 2-8 ℃,25 and 37℃for different times and after repeated freezing and thawing three times.Compared with the test results of each candidate standard product stored at-70 ℃ for a long time,the RSD were all within 5％.The collaborative results confirmed that the RSD of the polysaccharide content in different laborato-ries were less than 12％,of which assigned the concentration of four different serotype polysaccharide candidate standards.Conclusion:Serotypes 12F,14,17F and 22F polysaccharide standards have been developed,of which can be used for quality control of pneumococcal polysaccharide vaccines in the future.

Objective:To establish a TaqMan-MGB probe-based method for detecting the polymorphic loci C677T and A1298C of the MTHFR gene.Methods:Specific primers and TaqMan-MGB probes targeting the C677T and A1298C polymorphic loci of the MTHFR gene were designed and optimized based on the gene sequence information.A real-time quantitative PCR detection system was established.Gradient dilution experiments were conducted to determine the limit of detection,and reproducibility experiments were performed to evaluate detection consistency.Specificity was validated using wild-type and mutant plasmid templates.The method was applied to detect 56 clinical samples,and its accuracy and practicality were assessed through comparison with traditional Sanger sequencing.Results:The TaqMan-MGB probe method demonstrated high specificity for detecting the C677T and A1298C loci,with no cross-reactivity between wild-type and mutant probes,enabling accurate genotype differentiation.Sensitivity experiments revealed detection limits of 1.13 × 103 copies/μL for C677T and 8.39 × 101 copies/μL for A1298C.Reproducibility experiments showed coefficients of variation below 1％,indicating stable and reliable results.Among the 56 clinical samples,the overall detection rate for the C677T locus was 86.99％,and for the A1298C locus,it was 97.92％.The TaqMan-MGB method exhibited good concordance with Sanger sequencing results.Conclusion:The TaqMan-MGB method exhibits high specificity,sensitivity,and excellent reproducibility in detecting the polymorphic loci C677T and A1298C of the MTHFR gene,making it suitable for rapid detection in large-scale clinical samples.This method provides an effective molecular diagnostic tool for the early diagnosis and prevention of folate-related diseases.

Objective:To explore the intervention effects of the skill training for parents with autism child (STPAC) on toddlers with autism spectrum disorder (ASD).Methods:A multicenter non-randomized concurrent controlled study design was conducted. Thirty children with ASD aged 15-30 months, first diagnosed at the Children′s Hospital of Fudan University, the First Hospital of Jilin University, and Chengdu Women′s and Children′s Central Hospital from 2019 to 2020, were enrolled in the STPAC group. Thirty children with ASD who visited the same hospitals during the same period but refused the STPAC intervention were selected as the control group. The STPAC group received an 8-week intervention (3 h/week) followed by quarterly follow-ups for 1 year, while the control group voluntarily chose community-based routine interventions. The Griffiths development scales-Chinese (GDS-C) was used to assess the developmental levels, and the communication and symbolic behavior scales developmental profile infant-toddler checklist (CSBS-DP-ITC) was completed by the primary caregivers to evaluate social, language and symbolic behavior. The independent samples t-tests or Mann-Whitney U tests, etc.was used for inter-group comparison. The paired t-tests or Wilcoxon signed-rank tests, etc. was used for inter-group pre-post intervention comparison. Results:The STPAC group included 30 children (22 males and 8 females, aged (23.9±2.2) months), and the control group included 30 children (20 males and 10 females, aged (24.2±2.6) months). Before the intervention, there were no statistically differences in GDS-C development quotient (DQ) and CSBS-DP-ITC scores between groups (all P>0.05). After 1-year intervention, GDS-C DQ in personal-social, hearing-language, hand-eye coordination, performance domains of STPAC group and GDS-C DQ in personal-social, hearing-language domains of control group were all increased (all P<0.01). After 1-year intervention, CSBS-DP-ITC scores of both groups were all improved in socia, language, symbolic behavior, and total scores (all P<0.001). GDS-C DQ changes before and after 1 year of intervention in hearing-language, hand-eye coordination, performance domains of the STPAC group were all higher the those of control group (34(15, 48 vs. 10(-4, 39), 11±20 vs. -1±19, 23±25 vs. 8±22, all P<0.05). CSBS-DP-ITC scores changes before and after 1 year of intervention in social and total scores of the STPAC group were both higher the those of control group (10(5, 30) vs. 3(1, 7), 26±17 vs. 11±8, both P<0.001). Conclusion:Compared with the community routine interventions, the STPAC better improves the language, hand-eye coordination, visual-spatial, social communication, and play skills in ASD toddlers.