ObjectiveTo construct a risk prediction model for frequent acute exacerbations of chronic obstructive pulmonary disease （COPD） under disease-syndrome combination， thus providing decision support for precise clinical intervention. MethodsA total of 2 029 patients with acute exacerbations of COPD admitted to the First Affiliated Hospital of Anhui University of Chinese Medicine from January 2020 to August 2024 were retrospectively included. These patients were classified into groups of frequent acute exacerbations （≥2 times/year） and infrequent acute exacerbations （<2 times/year） according to the hospitalization times per year. Risk factors were screened by LASSO regression combined with logistic regression， and a nomogram model was constructed. The model performance was assessed based on the area under the curve （AUC）， calibration curves， and decision curve analysis （DCA）. ResultsThe differences in baseline characteristics between the frequent acute exacerbations group （1 196 cases） and infrequent acute exacerbations group （833 cases） were not statistically significant. LASSO regression combined with multivariate logistic regression screened the following independent risk factors： body mass index （BMI）， hospitalization days， number of smoking years， place of residence， use of noninvasive ventilators， oxygen-demanding therapy， liver cirrhosis， use of systemic glucocorticosteroids， and traditional Chinese medicine syndrome （phlegm and stasis obstructing the lung）. The nomogram model showed good discrimination and calibration in both the training set （AUC=0.748） and validation set （AUC=0.774）. ConclusionThe risk prediction model for frequent acute exacerbations of COPD， integrating traditional Chinese medicine syndrome， constructed in this study has high accuracy. It can provide a scientific basis for early clinical identification of high-risk patients and individualized intervention.

ObjectiveTo investigate the effects of hypoxia-treated mesenchymal stem cell （MSCs） exosomes （Exo） and their loading with curcumin on microglial inflammatory responses， and to explore the enhancing effect of hypoxia treatment on the function of MSCs Exo. MethodsThe supernatants of human umbilical cord （hUC）-MSCs cultured under normal and hypoxic conditions were collected， and Exo were isolated using ultracentrifugation. After identification by transmission electron microscopy and Western blot， curcumin was loaded using the co-incubation method. The lipopolysaccharide （LPS）-induced microglial inflammation model was treated with dimethyl sulfoxide （DMSO）， curcumin， normoxia Exo， hypoxia Exo， normoxic Exo loaded with curcumin， and hypoxic Exo loaded with curcumin， respectively. The expression of the M1-type marker inducible nitric oxide synthase （iNOS） in BV2 cells was detected by immunofluorescence （IF）. Western blot and enzyme-linked immunosorbent assay （ELISA） were used to measure the expression and secretion levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and IL-6 in the cells and their culture supernatants. ResultsNormoxia Exo， hypoxia Exo， normoxic Exo loaded with curcumin， and hypoxic Exo loaded with curcumin exhibited a "saucer-like" shape with a diameter ranging from 30~150 nm， and the expression of exosomal markers CD9， CD81， and TSG101 were positive. After treating the BV2 cell inflammation model， IF results showed that， compared with the normoxia Exo group， treatment with hypoxic Exo significantly reduced the expression of iNOS. Moreover， when compared with the curcumin group and the normoxic Exo loaded with curcumin group， the expression level of iNOS significantly decreased after treatment with hypoxic Exo loaded with curcumin. The results of Western blot and ELISA indicated that， in comparison with the normoxia Exo group， treatment with hypoxic Exo significantly reduced the expression and secretion of the inflammatory cytokines TNF-α， IL-1β， and IL-6. Additionally， when compared with the curcumin group and the normoxic Exo loaded with curcumin group， both the expression and secretion of TNF-α， IL-1β， and IL-6 significantly decreased after treatment with hypoxic Exo loaded with curcumin. ConclusionHypoxia preconditioning can enhance the ability of hUC-MSCs-Exo in the inhibition of microglial polarization and inflammatory factors’ secretion. Additionally， using Hypoxia-MSCs-Exo as a drug-delivery carrier of curcumin can improve its solubility and stability， facilitating its absorption by cells and exerting the therapeutic effect of combination therapy.

ObjectiveTo investigate the protective effect of liver-targeted fibroblast growth factor 21 （FGF21） overexpression against CCl₄-induced acute liver injury （ALI） and its association with silent information regulator 1/glutathione peroxidase 4 （SIRT1/GPX4） expression and ferroptosis-related markers. MethodsFemale ICR mice （8 weeks old） were randomly divided into Control， CCl₄， adeno-associated virus （AAV）-FGF21， and AAV-FGF21 + CCl₄ groups. Liver-targeted FGF21 overexpression was achieved via tail vein injection of AAV8-FGF21 virus with green fluorescent protein （GFP） labeling. ALI was induced by intraperitoneal injection of CCl₄ （0.3 mL/kg）， with sample collection at 12 hours. Body weight and liver weight were recorded to calculate the liver weight/body weight. Alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels were measured using a biochemical analyzer. FGF21 expression in liver tissues was detected by immunofluorescence. Hepatic histopathology was evaluated by HE staining. RT-qPCR was performed to assess mRNA expression of ferroptosis and iron metabolism-related genes （ACSL4， PTGS2， HJV， FPN， GPX4）， FGF21， and SIRT1. Protein expression of FGF21， SIRT1， and GPX4 were determined by Western blot. Iron content， malondialdehyde （MDA）， glutathione （GSH）， and superoxide dismutase （SOD） activity in liver homogenate were quantified using assay kits. ResultsCompared with the Control group， the FGF21 mRNA and protein expression elevated in CCl₄ group； serum ALT/AST levels and liver weight/body weigh increased. The HE staining results showed that the liver cell membranes were ruptured. The mRNA levels of ACSL4 and PTGS2， the iron content and MDA levels all increased （P<0.01）. The mRNA levels of SIRT1， GPX4， HJV， and FPN， the GSH level and SOD activity all decreased （P<0.05）， and the protein levels of SIRT1 and GPX4 both decreased （P<0.05）. Compared with the CCl₄ group， the serum levels of ALT and AST in the AAV-FGF21 + CCl4 group were decreased （P<0.01）， and the liver/body ratio of mice decreased （P<0.01）. The results of HE staining showed that the degree of liver injury was alleviated. The mRNA levels of ACSL4 and PTGS2， iron content and MDA level all decreased （P<0.01）， while the mRNA levels of SIRT1， GPX4， HJV， FPN， GSH level and SOD activity all increased （P<0.05）. The protein levels of SIRT1 and GPX4 both increased （P<0.05）. ConclusionFGF21 overexpression is associated with enhanced SIRT1/GPX4 expression and suppression of ferroptosis features， thereby contributing to the alleviation of CCl₄-induced ALI.

ObjectiveTo evaluate the clinical efficacy and safety of Wenshen Yangxue decoction combined with endometrial microstimulation in the treatment of infertile patients with polycystic ovary syndrome （PCOS， syndrome of kidney-Yang deficiency and blood stasis）. MethodsA randomized， positive-drug parallel-controlled， open-label clinical study was conducted. A total of 240 infertile PCOS patients with the syndrome of kidney-Yang deficiency and blood stasis who met the inclusion and exclusion criteria were randomly assigned into 4 groups， with 60 patients in each group. The control group was treated with clomiphene citrate （50-100 mg·d^-¹， orally for 5-10 consecutive days） for ovulation induction. On the basis of the therapy in the control group， the Wenshen group was additionally treated with Wenshen Yangxue decoction （250 mL， warm oral administration， twice daily， in the morning and evening）， and the stimulation group received endometrial microstimulation during the early follicular phase. The combined group was treated with clomiphene citrate plus Wenshen Yangxue decoction （at the same dosages as aforementioned） and endometrial microstimulation. The course of treatment for all the groups was 3 menstrual cycles. The pregnancy rate， ovulation status， sex hormone levels， glucose metabolism indicators， coagulation function， endometrial thickness， traditional Chinese medicine （TCM） symptom scores， and adverse reactions were observed in each group. ResultsA total of 239 patients were included in the final analysis， with 59 patients in the Wenshen group and 60 patients in each of the other three groups. The pregnancy rates were 43.3%（26/60） in the combined group and 32.2%（19/60） in the Wenshen group， both significantly higher than that （10%） in the control group （χ²=17.56，P<0.01）. Compared with the control group， the combined and Wenshen groups had increased mature follicle rates （P<0.01）， while the Wenshen group showed a decreased luteinization rate （P<0.05）. All the groups exhibited significant reductions in TCM symptom scores after treatment， and the Wenshen and combined groups had lower scores than the control group （P<0.01）. No statistically significant difference was found in the incidence of adverse events among the four groups， and all the adverse events were mild. ConclusionWenshen Yangxue decoction combined with endometrial microstimulation can alleviate the TCM symptoms of infertile PCOS patients with the syndrome of kidney-Yang deficiency and blood stasis， promote follicular maturation and ovulation， increase the cumulative pregnancy rate， and has good safety. It can provide new clinical evidence for the integrated traditional Chinese and Western medicine treatment of PCOS infertility.

ObjectiveTo evaluate the clinical efficacy and safety of Wenshen Yangxue decoction combined with endometrial microstimulation in the treatment of infertile patients with polycystic ovary syndrome （PCOS， syndrome of kidney-Yang deficiency and blood stasis）. MethodsA randomized， positive-drug parallel-controlled， open-label clinical study was conducted. A total of 240 infertile PCOS patients with the syndrome of kidney-Yang deficiency and blood stasis who met the inclusion and exclusion criteria were randomly assigned into 4 groups， with 60 patients in each group. The control group was treated with clomiphene citrate （50-100 mg·d^-¹， orally for 5-10 consecutive days） for ovulation induction. On the basis of the therapy in the control group， the Wenshen group was additionally treated with Wenshen Yangxue decoction （250 mL， warm oral administration， twice daily， in the morning and evening）， and the stimulation group received endometrial microstimulation during the early follicular phase. The combined group was treated with clomiphene citrate plus Wenshen Yangxue decoction （at the same dosages as aforementioned） and endometrial microstimulation. The course of treatment for all the groups was 3 menstrual cycles. The pregnancy rate， ovulation status， sex hormone levels， glucose metabolism indicators， coagulation function， endometrial thickness， traditional Chinese medicine （TCM） symptom scores， and adverse reactions were observed in each group. ResultsA total of 239 patients were included in the final analysis， with 59 patients in the Wenshen group and 60 patients in each of the other three groups. The pregnancy rates were 43.3%（26/60） in the combined group and 32.2%（19/60） in the Wenshen group， both significantly higher than that （10%） in the control group （χ²=17.56，P<0.01）. Compared with the control group， the combined and Wenshen groups had increased mature follicle rates （P<0.01）， while the Wenshen group showed a decreased luteinization rate （P<0.05）. All the groups exhibited significant reductions in TCM symptom scores after treatment， and the Wenshen and combined groups had lower scores than the control group （P<0.01）. No statistically significant difference was found in the incidence of adverse events among the four groups， and all the adverse events were mild. ConclusionWenshen Yangxue decoction combined with endometrial microstimulation can alleviate the TCM symptoms of infertile PCOS patients with the syndrome of kidney-Yang deficiency and blood stasis， promote follicular maturation and ovulation， increase the cumulative pregnancy rate， and has good safety. It can provide new clinical evidence for the integrated traditional Chinese and Western medicine treatment of PCOS infertility.

Objective : To investigate the protective effect of dimethyl fumarate(DMF) on maternal intrahepatic cholestasis(ICP) during pregnancy induced by di(2-ethylhexyl) phthalate(DEHP) exposure and its mechanism. Methods : Thirty-two 8-week-old female institute of cancer research(ICR) mice were randomly divided into 4 groups: Ctrl group, DEHP group, DMF group and DEHP+DMF group. DEHP and DEHP+DMF groups were treated with DEHP(200 mg/kg) by gavage every morning at 9:00 a.m. DMF and DEHP+DMF groups were treated with DMF(150 mg/kg) from day 13 to day 16 of gestation by gavage. After completion of gavage on day 16 of pregnancy, maternal blood, maternal liver, placenta, and amniotic fluid were collected from pregnant mice after a six-hour abrosia. The body weight of the mother rats and the body weight of the fetus rats were sorted and analyzed; the levels of total bile acid(TBA), alkaline phosphatase(ALP), aspartate aminotransferase/alanine aminotransferase(AST/ALT) in serum and TBA in liver, amniotic fluid and placenta were detected by biochemical analyzer; HE staining was used to observe the pathological changes of liver tissue; Quantitative reverse transcription PCR(RT-qPCR) was used to detect the expression levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-1, IL-18 and NOD-like receptor thermal protein domain associated protein 3(NLRP3) in the liver; Western blot was used to detect the expression of the nuclear factor KappaB(NF-κB) and NLRP3. Results : Compared with the control group, the body weight of the DEHP-treated dams and pups decreased(P<0.05); the levels of TBA, ALP, AST/ALT in the serum of dams and the levels of TBA in the liver, amniotic fluid, and placenta of dams increased(P<0.05); the histopathological results showed that liver tissue was damaged, bile ducts were deformed, and there was inflammatory cell infiltration around them; the levels of inflammation-related factors TNF-α, IL-6, IL-1, IL-18 and NLRP3 transcription in maternal liver increased(P<0.05); the expression of NF-κB and NLRP3 protein in maternal liver significantly increased( P<0. 05). Compared with the DEHP group,the body weight of both dams and fetuses significantly increased in DEHP + DMF group( P<0. 05); the levels of TBA,ALP,AST/ALT in the serum of dams and amniotic fluid of fetuses decreased( P<0. 05); the degree of liver lesions was improved; the transcription levels of inflammation-related factors TNF-α,IL-6,IL-1,IL-18 and NLRP3 in maternal liver decreased( P<0. 05); the expression of NF-κB and NLRP3 protein in maternal liver significantly decreased( P<0. 05). Conclusion DMF can effectively protect the DEHP exposure to lead to female ICP,and its mechanism may be through inhibiting the NF-κB/NLRP3 pathway and reducing liver inflammation.

OBJECTIVE: To analyze the sequencing results, protein structure model, and impact of mutations on the dynamic stability of glycosyltransferase (GTA) in a case with Aw26 blood group subtype. METHODS: ABO phenotype was determined by serological testing (anti-A, anti-B, anti-H, and reverse typing). Potential variant of the ABO gene was identified by Sanger sequencing, and the haploid sequence of the variant site was analyzed by TOPOT-A cloning. Molecular models of the GTA was generated by PyMol, and 100-ns molecular dynamics (MD) was simulated with GROMACS software to assess the conformational stability using root mean square deviation (RMSD), radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding, and binding free energy. RESULTS: Serological assays confirmed the proband as an Aw subtype, whose genotype was identified as ABO*Aw.26/ABO*O.01.02 with variants including p.Pro156Leu, p.Arg176His and p.Pro354ArgfsTer23. Haploid sequencing validated the results of direct sequencing. Molecular modeling showed that the p.Arg176His variant could reduce water-mediated hydrogen bonds from six (wild-type) to one (variant). MD simulation revealed the wild type system could achieve equilibrium within 10 ns (mean RMSD ≈ 0.30 nm), whilst the mutant system required 50 ns to equilibrate and exhibited greater fluctuation (mean RMSD ≈ 0.40 nm). Root mean square fluctuation (RMSF) analysis confirmed significantly increased flexibility in the mutant's N-terminal loop (residues 63-76). The mutant Rg displayed an expansion-contraction transition within 0 ~ 40 ns, and its SASA value has increased. The number of hydrogen bonds and binding energy of the mutant had decreased (wild-type: 5 to 8, binding energy: -11.53 kcal/mol; mutant: 2 to 5, binding energy:-8.52 kcal/mol). CONCLUSION An Aw26 subtype was identified. The p.Arg176His and p.Pro354Argfs*23p variants could synergistically compromise the structural stability of GTA and its substrate binding capacity by disrupting the hydrogen-bond network, increasing local flexibility, and reducing the overall conformational stability.
ABO Blood-Group System/chemistry* ; Humans ; Molecular Dynamics Simulation ; Models, Molecular ; Mutation ; Genotype ; Protein Conformation ; Glycosyltransferases/chemistry* ; Male

Objective To explore the effect of acyl-CoA synthetase short-chain family member 3(ACSS3)gene on the proliferation of human large cell lung cancer cells(NCI-H460)using CRISPR/Cas 9 gene editing technology.Methods The expression of ACSS3 was detected by Western blot.ACSS3-targeting sgRNAs were designed,and a CRISPR/Cas 9 knockout vector was constructed and transfected into NCI-H460 cells.The transfected cells were selected with puromycin based on vector-carried resistance.ACSS3-knockout monoclonal cell strains were established by limited dilution method and then expanded in culture.Knockout efficiency was confirmed by Western blot.Cell proliferation was assessed using MTT and colony formation assays.Results The expression of ACSS3 was significantly elevated in NCI-H460 cells as compared with human normal lung epithelial cells BEAS-2B(P＜0.05).No ACSS3 protein was detected in the knockout monoclonal strain,indicating successful generation of ACSS3-knockout NCI-H460 cells.Compared with the control cells transfected with empty vector,the proliferation and colo-ny formation ability were inhibited in NCI-H460 cells with ACSS3 knockout(P＜0.05).Conclusions The ACSS3-knockout NCI-H460 cell strain was successfully established,which provides a foundation for further study on the role of ACSS3 in lung cancer.

OBJECTIVE: To observe the effect of acupuncture at "pelvic floor six needles" for mild to moderate female stress urinary incontinence (SUI). METHODS: A total of 60 patients with mild to moderate female SUI were randomly divided into an observation group and a control group, 30 cases each group. The control group received Kegel exercise. The observation group received acupuncture at "pelvic floor six needles" on the basis of the treatment as the control group, bilateral Zhongliao (BL33), Zhibian (BL54), Huiyang (BL35), Shuidao (ST28), Dahe (KI12) and Guanyuan (CV4) were selected, once every other day, 3 times a week, 4 weeks as a course of treatment, a total of 2 courses were required. Before treatment and after 4, 8 weeks of treatment, urine leakage in 1 hour, International Consultation on Incontinence questionnaire short form (ICI-Q-SF) score, and incontinence quality of life questionnaire (I-QOL) score were observed in the two groups, and the clinical efficacy was evaluated. RESULTS: After 8 weeks of treatment, urine leakage in 1 hour and ICI-Q-SF scores in both groups were decreased compared with those before treatment (P<0.05), and urine leakage in 1 hour and ICI-Q-SF score in the observation group were lower than those in the control group (P<0.05). After 4, 8 weeks of treatment, I-QOL scores were increased compared with those before treatment in both groups (P<0.05), and the I-QOL scores in the observation group were higher than those in the control group (P<0.01, P<0.001). The total effective rate of the observation group was 93.3% (28/30), which was higher than 73.3% (22/30) in the control group (P<0.05). CONCLUSION Acupuncture at "pelvic floor six needles" could improve the clinical symptoms and quality of life in patients with mild to moderate female SUI to a certain degree.
Humans ; Female ; Acupuncture Therapy/instrumentation* ; Urinary Incontinence, Stress/physiopathology* ; Middle Aged ; Adult ; Exercise Therapy ; Acupuncture Points ; Pelvic Floor/physiopathology* ; Aged ; Treatment Outcome ; Quality of Life

OBJECTIVE: To observe the effects of Xingnao Kaiqiao acupuncture technique (for regaining consciousness and opening orifice) on methylation of N6-methyladenosine (m6A), and key methyltransferases and demethylases, so as to clarify the mechanism of acupuncture on cerebral ischemia-reperfusion injury (CIRI). METHODS: Of 68 male Sprague-Dawley rats of SPF grade, 15 rats were randomly selected as a sham-operation group, and the remaining rats were subjected to the model of middle cerebral artery occlusion using the suture ligation. CIRI was induced by ischemia for 2 h followed by reperfusion. Rats that failed to modeling or died were excluded. The rest 45 rats were randomly divided into three groups, i.e. model group, acupuncture group, and non-point acupuncture group, with 15 rats in each group. The rats in the acupuncture group were treated with acupuncture at bilateral "Neiguan" (PC6) and "Shuigou" (GV26). In the non-point acupuncture group, acupuncture was delivered at three non-points, located 3 mm below the bilateral midaxillary line and 3 mm lateral to the tip of the coccyx. One intervention was operated in these two acupuncture groups and the needles were retained for 30 min. Before modeling start and 2 h after ischemia, a laser speckle flowmeter was used to monitor the cerebral blood perfusion. In 2 h of ischemia and 24 h of reperfusion, the neurological behavioral score was evaluated. The volume of rat cerebral infarction was determined by triphenyltetrazolium chloride (TTC) staining, and the level of m6A methylation in ischemic cortical area was detected by Dot blot, and the protein and mRNA expression of the demethylase i.e. fat mass and obesity-associated protein (FTO), AlkB homolog 5 (ALKBH5) and key methyltransferases, i.e. methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associated protein (WTAP) in ischemic cortical area were detected by Western blot and real-time PCR. RESULTS: Cerebral blood perfusion decreased by>70% after 2 h ischemia. Compared with the sham-operation group, the neurobehavioral score and the percentage of cerebral infarction volume increased in the model group (P<0.01); the level of m6A methylation in the ischemic cortical area increased (P<0.01), the protein and mRNA expression of FTO decreased (P<0.01), and that of ALKBH5, METTL3, and METTL14 increased (P<0.01, P<0.05) in the model group. When compared with the model group and the non-point acupuncture group, the acupuncture group showed a decrease in the neurobehavioral score and the percentage of cerebral infarction volume (P<0.01), the level of m6A methylation in the ischemic cortical area was reduced (P<0.01, P<0.05), and the protein and mRNA expression of FTO was elevated (P<0.01). CONCLUSION Xingnao Kaiqiao acupuncture technique presents its protective effect on the brain in the rats with CIRI, which is related to up-regulating the expression of FTO and modulating m6A methylation.
Animals ; Rats, Sprague-Dawley ; Male ; Acupuncture Therapy ; Reperfusion Injury/genetics* ; Rats ; Brain Ischemia/genetics* ; Humans ; Adenosine/metabolism* ; Methylation ; Acupuncture Points ; Cerebral Cortex/metabolism*