ObjectiveThis study aims to explore the mechanism of action of Huangqi Guizhi Wuwutang in treating rheumatoid arthritis （RA）-associated sarcopenia by regulating autophagy and improving skeletal muscle homeostasis based on network pharmacology，bioinformatics，machine learning，and animal experiments. MethodsActive ingredients and targets of Huangqi Guizhi Wuwutang were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP），PubChem，and SwissTargetPrediction databases. RA-related datasets were retrieved from the GEO database，and differential genes were screened. Sarcopenia-related targets were searched through GeneCards and the Comparative Toxicology Database （CTD），and autophagy-related gene sets were downloaded from the Human Autophagy Database （HADb）. Their intersection was analyzed to identify autophagy-related therapeutic targets，followed by enrichment analysis. A protein-protein interaction （PPI） network was constructed using the STRING database，and key targets were selected using multiple methods. Machine learning was applied to predict models based on the expression profiles of intersecting targets，and nomogram models were constructed based on key targets. Molecular docking of the top four active ingredients with key targets was performed using AutoDockVina. A collagen-induced arthritis （CIA） rat model was established using bovine type Ⅱ collagen，with SD rats divided into groups including a blank group，a model group，and low-，medium-，and high-dose groups of Huangqi Guizhi Wuwutang （2.44，4.88，and 9.76 g·kg^-1） and administered for five consecutive weeks. Joint scores and gastrocnemius muscle mass were recorded and analyzed after modeling. Hematoxylin and eosin （HE） staining and Masson's staining were used to observe pathological changes in muscle tissue. Immunofluorescence staining was applied to observe the protein expression levels of myosin heavy chain （MYHC） and insulin-like growth factor-1 （IGF-1） in skeletal muscle. Western blot was used to detect the protein expression levels of autophagy-related proteins ATG5，Beclin1，LC3B，muscle-specific proteins （MuRF1），MaFbx，and MYHC. Real-time quantitative reverse transcription PCR （Real-time PCR） was performed to measure the mRNA expression levels of ATG5，Beclin1，LC3B，MuRF1，MaFbx，and MYHC in muscle tissue. ResultsNetwork pharmacology revealed that Huangqi Guizhi Wuwutang shared 25 common targets with autophagy genes related to RA-associated sarcopenia. The PPI network and machine learning identified six key targets，which were primarily involved in autophagy and inflammatory pathways. Animal experiments showed that compared to the blank group，the model group had significantly higher joint scores （P<0.01） and lower gastrocnemius muscle index （P<0.01）. HE staining indicated a significant reduction in the cross-sectional area of gastrocnemius muscle fibers，with notable inflammatory cell infiltration and muscle atrophy in the model group. Masson's staining revealed obvious collagen fiber proliferation and deposition，with significant muscle fibrosis in the model group. The protein and mRNA expression levels of ATG5，Beclin1，LC3B，MuRF1，and MaFbx were significantly increased （P<0.01），while the protein expression of MYHC and IGF1 was significantly downregulated （P<0.01）. Compared with the model group，the high-dose group of Huangqi Guizhi Wuwutang showed significantly reduced protein and mRNA expression levels of ATG5，Beclin1，LC3B，MuRF1，and MaFbx （P<0.01） and increased protein expression levels of MYHC and IGF1 （P<0.01）. The cross-sectional area of muscle fibers increased，and the muscle cell morphology approached normal. Moreover，pathological abnormalities in the gastrocnemius muscle were significantly improved，with reduced collagen fiber proliferation （P<0.01）. ConclusionHuangqi Guizhi Wuwutang can mediate autophagy by regulating the expression of ATG5，Beclin1，LC3B，and IGF1，thereby reducing skeletal muscle catabolism and improving skeletal muscle homeostasis，which contributes to the treatment of RA-associated sarcopenia. The findings provide insight into the mechanisms underlying the effects of Huangqi Guizhi Wuwutang in the treatment of RA-related sarcopenia and offer a reference for its enhanced clinical application.

Objective To investigate the effect and mechanisms of celecoxib on right heart function in mice with acute high-altitude hypoxia exposure.Methods Male C57BL/6J mice(7 weeks old)were housed in a hypobaric chamber simulating an altitude of 5 800 m for 2 d to establish an animal model of acute hypobaric hypoxia.①Eighteen mice were randomly assigned to plain+saline(P+S),high-altitude hypoxia exposure+saline(H+S),and high-altitude hypoxia exposure+celecoxib(H+Cel).Body weight and routine blood indicators were measured,and cardiac ultrasound examination were performed for heart rate(HR),pulmonary artery acceleration time to ejection time ratio(AT/ET),tricuspid annular plane systolic excursion(TAPSE),tricuspid annular systolic velocity(S'),and left ventricular ejection fraction(LVEF)and fractional shortening(FS).Targeted metabolomic profiling was applied to detect the cardiac arachidonic acid(AA)metabolite levels.The contents of 12,13-dihydroxy-9Z-octadecenoic acid(12,13-diHOME)in the heart,liver,brown adipose tissue,and plasma were quantified by ELISA.② Eighteen mice were randomly assigned into plain+saline(P+S),high-altitude hypoxia exposure+saline(H+S)and high-altitude hypoxia exposure+12,13-diHOME(H+di)groups.Body weight,routine blood tests,and echocardiography were performed as above.③ Thirty-two mice were randomly divided into high-altitude hypoxia exposure+saline(H+S),high-altitude hypoxia exposure+celecoxib(H+Cel),high-altitude hypoxia exposure+soluble epoxide hydrolase inhibitor(sEHI)(H+sEHI),and high-altitude hypoxia exposure+sEHI+celecoxib(H+sEHI+Cel)groups.Body weight,routine blood tests,and echocardiography were performed as above.Cardiac and plasma contents of 12,13-diHOME and epoxyeicosatrienoic acids(EETs)were measured by ELISA.Results ① Compared to the P+S group,the H+S group exhibited significantly reduction of cardiac 12,13-diHOME level(P<0.001),increased counts of white blood cells(WBC)and neutrophils(P<0.01)and decreased TAPSE,S'and AT/ET both at resting state and under stress(P<0.01,P<0.001).Compared to the H+S group,the H+Cel group exhibited significantly increase of cardiac 12,13-diHOME level(P<0.05),reduced WBC and lymphocyte counts(P<0.01,P<0.05)and improved TAPSE and S'levels at resting state and under stress(P<0.01,P<0.001).② Compared to the H+S group,the H+di group demonstrated significantly improvement of TAPSE at basal and under stress(P<0.001)and a trend towards improved TAPSE at resting state(P=0.0532),but no obvious differences was observed in WBC and neutrophil counts between the H+di group and the H+S group.③ Compared to the H+Cel group,both the H+sEHI and H+sEHI+Cel groups exhibited significantly reduction of cardiac 12,13-diHOME level(P<0.01,P<0.05)though no statistical changes in cardiac function indicators.Compared to the H+S group,WBC counts and lymphocyte were decreased,and serum EETs level was incrased in the H+Cel group,H+sEHI group and H+sEHI+Cel group(P<0.01,P<0.001).Conclusion Celecoxib can elevate cardiac level of 12,13-diHOME and improves right heart function in mice after acute high-altitude hypoxia exposure through the CYP450-sEH metabolic pathway.

Objective:To investigate whether changes in CT pulmonary angiography (CTPA) parameters before and after balloon pulmonary angioplasty (BPA) are correlated with treatment effects in patients with chronic thromboembolic pulmonary hypertension (CTEPH).Methods:A retrospective study was conducted, including patients with CTEPH who underwent BPA treatment at Beijing Anzhen Hospital from November 2021 to Febbruary 2024. Clinical data and CTPA parameters were analyzed before the initial BPA session and at least 6 months after the final BPA session. Clinical data included WHO functional class, 6-minute walk distance (6MWD), and plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels. The CTPA parameters included the widest diameter of the pulmonary artery diameter (dPA), ascending aorta diameter (dAA), the widest transverse diameter of the right atrium (dRA), the widest short-axis diameter of the right ventricle (dRV), the widest short-axis diameter of the left ventricle (dLV), and the diameter of right ventricular free wall thickness(dRVW). Multiple linear regression was applied to identify variables associated with the decrease in mean pulmonary artery pressure (mPAP) among the CTPA parameters.Results:All CTEPH patients underwent a total of 115 BPA treatments. Postoperatively, the WHO functional class of the patients improved compared to preoperative levels ( χ2=5.01, P<0.001), 6MWD improved ( t=4.50, P<0.001), and NT-proBNP levels decreased ( Z=4.47, P<0.001). Hemodynamic parameters, including mPAP, pulmonary vascular resistance, cardiac output, and cardiac index, were significantly improved postoperatively (all P<0.001). CTPA-related parameters, including dPA, dRA, dRV, dRVW, dPA/dAA, dRV/dLV, and dRA/dPA, significantly decreased compared to preoperative values (all P<0.001). Multiple linear regression analysis showed that the decrease in dPA ( β=0.314, P=0.037) and dRA ( β=0.334, P=0.046) were significantly correlated with the improvement in mPAP. Conclusions:Exercise tolerance, hemodynamics, and CTPA parameters in patients with CTEPH significantly improved after BPA treatment. The decrease in dPA and dRA were significantly correlated with the improvement in mPAP, suggesting that CTPA is a potentially novel, objective, effective, and noninvasive method for evaluating the therapeutic efficacy of BPA.

Objective:To investigate whether changes in CT pulmonary angiography (CTPA) parameters before and after balloon pulmonary angioplasty (BPA) are correlated with treatment effects in patients with chronic thromboembolic pulmonary hypertension (CTEPH).Methods:A retrospective study was conducted, including patients with CTEPH who underwent BPA treatment at Beijing Anzhen Hospital from November 2021 to Febbruary 2024. Clinical data and CTPA parameters were analyzed before the initial BPA session and at least 6 months after the final BPA session. Clinical data included WHO functional class, 6-minute walk distance (6MWD), and plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels. The CTPA parameters included the widest diameter of the pulmonary artery diameter (dPA), ascending aorta diameter (dAA), the widest transverse diameter of the right atrium (dRA), the widest short-axis diameter of the right ventricle (dRV), the widest short-axis diameter of the left ventricle (dLV), and the diameter of right ventricular free wall thickness(dRVW). Multiple linear regression was applied to identify variables associated with the decrease in mean pulmonary artery pressure (mPAP) among the CTPA parameters.Results:All CTEPH patients underwent a total of 115 BPA treatments. Postoperatively, the WHO functional class of the patients improved compared to preoperative levels ( χ2=5.01, P<0.001), 6MWD improved ( t=4.50, P<0.001), and NT-proBNP levels decreased ( Z=4.47, P<0.001). Hemodynamic parameters, including mPAP, pulmonary vascular resistance, cardiac output, and cardiac index, were significantly improved postoperatively (all P<0.001). CTPA-related parameters, including dPA, dRA, dRV, dRVW, dPA/dAA, dRV/dLV, and dRA/dPA, significantly decreased compared to preoperative values (all P<0.001). Multiple linear regression analysis showed that the decrease in dPA ( β=0.314, P=0.037) and dRA ( β=0.334, P=0.046) were significantly correlated with the improvement in mPAP. Conclusions:Exercise tolerance, hemodynamics, and CTPA parameters in patients with CTEPH significantly improved after BPA treatment. The decrease in dPA and dRA were significantly correlated with the improvement in mPAP, suggesting that CTPA is a potentially novel, objective, effective, and noninvasive method for evaluating the therapeutic efficacy of BPA.

Lipophosphatidic acid(LTA)was used to stimulate Mac-T cells,and the expression lev-els and phosphorylation levels of key proteins of nuclear factor-κB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathway and the expression levels of upstream key action factors TLR4 and MyD88 proteins were detected by Western blot,and EDU assay was used to detect cell proliferation levels and flow cytometry was used to detect apoptosis.The results showed that acti-vation of GPR120 significantly decreased the phosphorylation levels of LTA-induced NF-κB(P65 and IκBα)(P＜0.01)and MAPK(JNK,ERK,p38)(P＜0.01)in Mac-T cells;inhibition of GPR120 was able to upregulate LTA-induced NF-κB(p65 and IκBα)in Mac-T cells(P＜0.01)and MAPK(JNK,ERK,p38)phosphorylation levels(P＜0.01);and activation of GPR120 significantly allevia-ted LTA-induced upregulation of TLR4 and MyD88(P＜0.01);inhibition of GPR120 significantly exacerbated LTA-induced upregulation of TLR4 and MyD88(P＜0.05);LTA stimulation led to a trend of diminished Mac-T cell proliferation and significantly increased apoptosis,whereas activa-tion of the GPR120 gene significantly increased cell activity(P＜0.01),promoted cell proliferation and significantly reduced apoptosis(P＜0.05)thereby alleviating the damage to Mac-T cells by LTA;LTA stimulation led to a highly significant increase in apoptosis(P＜0.01).In contrast,acti-vation of the GPR120 gene significantly reversed the increase in the apoptosis rate of Mac-T cells induced by LTA(P＜0.01),while inhibition of the GPR120 gene enhanced the apoptosis-promo-ting effect of LTA(P＜0.05),indicating that activation of the GPR120 gene attenuated the in-crease of apoptosis rate caused by LTA-induced inflammatory Mac-T cells.The results suggest that GPR120 can regulate inflammation by mediating TLR4 and MyD88 expression to inhibit NF-κB/MAPK inflammatory pathway activation and can promote cell proliferation.

Objective To explore whether hepatocyte Perilipin-2(Plin2)is involved in the development of fatty liver related to comparative gene identification-58(CGI-58)deficiency mice and compare the effects of Plin2 and Plin3 on lipid droplet formation and lipid accumulation.Methods Based on CGI-58Flox/Flox mice as animal model,the adeno-associated viruses targeting mouse liver,CGI-58 knockout and Plini2 knockdown were achieved by co-expression Cre protein and micro-RNA targeting Plin2(Mi-KD).Then CGI-58 deficiency mice were used as control(NC)to detect the differences in metabolic phenotype and liver pathology.AML-12 mouse hepatocytes were used as cellular model and interfered with siRNA to achieve Plin2/Plin3 knockdown in AML-12 cells.Lipid droplet formation and lipid accumulation were compared with Bodipy staining and enzyme colorimetry in basal condition or lipid-overloaded condition(OA inducement)after Plin2/Plin3 knockdown.Results Plin2 knockdown(Mi-KD)reduced PLIN2 protein level by＞99％in mouse livers.Mi-KD decreased hepatomegaly(P=0.019 5)and liver injury(P=0.000 4),while reduced the histological NAS score(P=0.000 2)and hepatic triglyceride content(P=0.016 6)in the CGI-58 deficiency female mice.Mi-KD prevented microvesicular hepatic steatosis in the CGI-58 deficient female mice.Plin3 knockdown significantly reduced the triglyceride content in basal condition of hepatocytes(P=0.001 4),and Plin2 knockdown just showed a decreased trend.Plin2 or Plin3 knockdown significantly reduced the triglyceride content separately in lipid-overloaded hepatocytes(P＜0.05).Conclusion Hepatocyte Plin2 is essential in the development of microvesicular hepatic steatosis caused by CGI-58 deficiency.Both Plin2 and Plin3 are involved in lipid droplet formation and lipid accumulation in hepatocytes,and Plin3 shows a stronger effect.

As a receptor protein,GPR120 is activated by long-chain fatty acids(such as omega-3 fat-ty acids,alpha-linolenic acid,eicosapentaenoic acid,and docosahexaenoic acid.It plays an important regulatory role in gastrointestinal peptide release,inflammation,lipogenesis,glucose tolerance,and insulin sensitivity.In order to study the synergistic regulation of the GPR120 gene on milk fat me-tabolism and its anti-inflammatory effects in dairy cow MAC-T cells,the GPR120 activator TUG-891 and the inhibitor AH7614 were used to treat both dairy cow MAC-T cells and LTA-induced inflammatory dairy cow MAC-T cells.This treatment aimed to detect the expression of key genes involved in milk fat synthesis and inflammatory factors.The results showed that the GPR120 gene activator significantly increased the relative expression levels of cholesterol regulatory element binding protein(SREBP1)and fatty acid synthase(FASN),key genes for milk fat synthesis.Addi-tionally,the expression levels of the inflammatory factor interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were reduced in the inflammatory dairy cow MAC-T cell model,which prelimi-natively reveals that GPR120 co-regulates milk fat and mammary inflammation in dairy cows,thereby laying a foundation for subsequent molecular mechanism research.

As a receptor protein,GPR120 is activated by long-chain fatty acids(such as omega-3 fat-ty acids,alpha-linolenic acid,eicosapentaenoic acid,and docosahexaenoic acid.It plays an important regulatory role in gastrointestinal peptide release,inflammation,lipogenesis,glucose tolerance,and insulin sensitivity.In order to study the synergistic regulation of the GPR120 gene on milk fat me-tabolism and its anti-inflammatory effects in dairy cow MAC-T cells,the GPR120 activator TUG-891 and the inhibitor AH7614 were used to treat both dairy cow MAC-T cells and LTA-induced inflammatory dairy cow MAC-T cells.This treatment aimed to detect the expression of key genes involved in milk fat synthesis and inflammatory factors.The results showed that the GPR120 gene activator significantly increased the relative expression levels of cholesterol regulatory element binding protein(SREBP1)and fatty acid synthase(FASN),key genes for milk fat synthesis.Addi-tionally,the expression levels of the inflammatory factor interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were reduced in the inflammatory dairy cow MAC-T cell model,which prelimi-natively reveals that GPR120 co-regulates milk fat and mammary inflammation in dairy cows,thereby laying a foundation for subsequent molecular mechanism research.

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). METHODS: A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. RESULTS: Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. CONCLUSION By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.
Humans ; Pregnancy ; Female ; Child ; Male ; Preimplantation Diagnosis ; East Asian People ; Semen ; Genetic Testing ; Muscular Atrophy, Spinal/genetics* ; Aneuploidy ; Blastocyst/pathology* ; High-Throughput Nucleotide Sequencing ; Spermatozoa

Objective:To construct a set of weighted trigger index system for dynamic adjustment of medical service price,so as to provide references and basis for price adjustment of complex medical service projects in Tangshan.Methods:The Delphi expert consultation method was used to demonstrate and determine the index system,and the difference comparison method and product method were used to determine the weight coefficient for each index.Results:The trigger index system of dynamic adjustment of medical service price including 4 first-level indicators,10 second-level indicators and 29 third-level indicators,and the weight coefficient of each indicator was given.Conclusion:The trigger index system of dynamic adjustment of medical service price in Tangshan has high scientificity and reliability,which is helpful for Tangshan to establish a sensitive and moderate price dynamic adjustment mechanism.Considering the affordability of all parties in the price adjustment of medical services in Tangshan,it is conducive to strengthening the total price control and structural adjustment of medical services in Tangshan.