ObjectiveTo investigate the mechanism through which miR‑21 improves chronic renal fibrosis in mice via targeted modulation of the phosphatase and tensin homolog （PTEN）/protein kinase B （AKT）/mammalian target of rapamycin （mTOR） pathway. MethodsThirty‑two chronic kidney disease model mice were randomly divided into four groups （n=8 each group）： model group， miR‑21 overexpression group， miR‑21 inhibition group， and miR‑21 inhibition + MK‑2206 group. Eight healthy mice were included as the control group. The miR‑21 overexpression， miR‑21 inhibition， and miR‑21 inhibition + MK‑2206 groups received tail‑vein injections of lentivirus （50 μL， 1×10⁸ TU per mouse） once weekly for three weeks. The control and model groups were injected with an equal volume of empty vector （LV‑NC）. The miR‑21 inhibition + MK‑2206 group additionally received gavage of the AKT/mTOR pathway inhibitor MK‑2206 （480 mg/kg） once weekly for three weeks. The expressions of miR‑21， 24 h urinary protein， serum creatinine （Scr）， blood urea nitrogen （BUN）， and renal tissue levels of collagen Ⅰ， collagen Ⅲ， α‑smooth muscle actin （α‑SMA）， and PTEN protein， as well as p‑AKT/AKT and p‑mTOR/mTOR ratios， were compared among groups. HE staining was used to observe pathological changes in renal tissue， and Masson staining was used to observe the degree of renal fibrosis. A dual‑luciferase assay was performed to verify the targeting relationship between miR‑21 and PTEN. ResultsCompared with the model group， miR‑21 expression in renal tissue increased in the miR‑21 overexpression group （P<0.05） and decreased in the miR‑21 inhibition group （P<0.05）. Compared with the model group， the miR‑21 overexpression group showed increased 24 h urinary protein， Scr， BUN， and renal tissue expression of collagen Ⅰ， collagen Ⅲ， and α‑SMA （all P<0.05）， while these indicators decreased in the miR‑21 inhibition group （P<0.05）. Compared with the miR‑21 inhibition group， the miR‑21 inhibition + MK‑2206 group exhibited lower 24‑h urinary protein， Scr， BUN， and renal tissue expression of Collagen Ⅰ， Collagen Ⅲ， and α‑SMA （all P<0.05）. Compared with the model group， the miR‑21 overexpression group showed decreased PTEN protein expression （P<0.05） and increased p‑AKT/AKT and p‑mTOR/mTOR ratios （P<0.05）， while the miR‑21 inhibition group showed increased PTEN expression （P<0.05） and decreased p‑AKT/AKT and p‑mTOR/mTOR ratios （P<0.05）. Compared with the miR‑21 inhibition group， the miR‑21 inhibition + MK‑2206 group had lower p‑AKT/AKT and p‑mTOR/mTOR ratios （P<0.05）， with no significant difference in PTEN protein expression. HE and Masson staining showed normal kidney structure and almost no fibrosis in the control group. The model group exhibited glomerular enlargement， capillary loop adhesion， and focal fibrosis. The miR-21 overexpression group showed severe destruction of glomerular structure， accompanied by extensive fibrosis and renal tubular atrophy. The pathological changes and degree of fibrosis were alleviated in the miR-21 inhibition group. The miR-21 inhibition + MK-2206 group showed only mild pathological changes and mild fibrosis， with the interstitium being largely normal. Compared with PTEN-WT + NC mimics 1， the relative luciferase activity in the PTEN-WT + miR-21 mimics group decreased （P<0.001）. There was no statistically significant difference in relative luciferase activity between PTEN-WT + NC mimics group and PTEN-MUT + miR-21 mimics group. ConclusionmiR‑21 may improve renal function indicators and alleviate renal fibrosis in chronic kidney disease mice via targeted modulation of PTEN and subsequently inhibiting the AKT/mTOR pathway.

ObjectiveTo investigate whether there exists an independent association beyond shared risk factors between colorectal space-occupying lesions and metabolic associated fatty liver disease （MAFLD）， and to provide new ideas for early identification of colorectal space-occupying lesions in the MAFLD population. MethodsA retrospective analysis was performed for 12 252 patients who were hospitalized in The First Affiliated Hospital of Shandong First Medical University （Shandong Provincial Qianfoshan Hospital） and underwent both colonoscopy and abdominal imaging （abdominal ultrasound， magnetic resonance imaging， or computed tomography） from September 1， 2018 to August 31， 2023. According to colonoscopy findings， the patients were divided into colorectal space-occupying lesion group with 6 545 patients and non-colorectal space-occupying lesion group with 5 707 patients. Baseline data were compared between the two groups. The univariate and multivariate Logistic regression analyses were used to identify influencing factors for colorectal space-occupying lesions， and further analysis was performed to investigate the association of MAFLD and liver fibrosis （assessed by fibrosis-4 ［FIB-4］， nonalcoholic fatty liver disease fibrosis score ［NFS］， and aspartate aminotransferase-to-platelet ratio index） with colorectal space-occupying lesions. The Mann-Whitney U test was used for comparison of continuous data with skewed distribution between groups， and the chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups. ResultsThe univariate analysis showed that compared with the non-colorectal space-occupying lesion group， the colorectal space-occupying lesion group had significantly higher age （Z=-95.628， P<0.001）， proportion of male patients （χ²=406.910， P<0.001）， body weight （Z=-24.928， P<0.001）， body mass index （BMI） （Z=-21.367， P<0.001）， diastolic blood pressure （Z=-15.527， P<0.001）， systolic blood pressure （Z=-16.965， P<0.001）， proportion of patients with history of coronary heart disease （χ²=28.112， P<0.001）/hypertension （χ²=152.993， P<0.001）/diabetes （χ²=52.175， P<0.001）/cerebral infarction （χ²=14.097， P<0.001）， white blood cell count （WBC） （Z=-12.801， P<0.001）， hemoglobin （HGB） （Z=-19.258， P<0.001）， aspartate aminotransferase （Z=-6.673， P=0.001）， alanine aminotransferase （Z=-10.375， P<0.001）， alkaline phosphatase （Z=-10.496， P<0.001）， gamma-glutamyl transpeptidase （GGT） （Z=-18.893， P<0.001）， blood urea nitrogen （Z=-13.291， P<0.001）， creatinine （Z=-16.798， P<0.001）， uric acid （Z=-16.822， P<0.001）， triglyceride （Z=-10.186， P<0.001）， and fasting blood glucose （Z=-15.761， P<0.001）. The multivariate Logistic regression analysis showed that age （odds ratio ［OR］=1.052， 95% confidence interval ［CI］： 1.045 — 1.057， P<0.001）， sex （OR=0.523， 95%CI： 0.466 — 0.587， P<0.001）， BMI （OR=1.031， 95%CI： 1.018 — 1.046， P<0.001）， history of hypertension （OR=1.140， 95%CI： 1.019 — 1.276， P=0.022）， WBC （OR=1.057， 95%CI： 1.028 — 1.087， P<0.001）， GGT （OR=1.001， 95%CI： 1.000 — 1.002， P=0.021）， Alb （OR=0.982， 95%CI： 0.969 — 0.995， P=0.008）， and HGB （OR=1.004， 95%CI： 1.001 — 1.008， P=0.019） were independent influencing factors for colorectal space-occupying lesions. In the MAFLD population， after adjustment for age， sex， BMI， history of hypertension， WBC， HGB， GGT， and Alb， intermediate-to-high risk of advanced liver fibrosis assessed by FIB-4 and NFS was still an influencing factor for colorectal space-occupying lesions （FIB-4： OR=1.457， 95%CI： 1.176 — 1.810， P<0.05； NFS： OR=1.499， 95%CI： 1.244 — 1.809， P<0.05）. Liver fibrosis degree based on FIB-4 was not significantly associated with the pathological type， location， size， or number of colorectal space-occupying lesions （all P>0.05）. While intermediate-to-high risk of advanced liver fibrosis based on NFS was not associated with pathological type， location， or size， it might be associated with the number of lesions （χ²=9.770， P<0.05）. ConclusionAge， sex， BMI， history of hypertension， WBC， HGB， GGT， and Alb are independent influencing factors for colorectal space-occupying lesions. Intermediate-to-high risk of advanced liver fibrosis assessed by FIB-4 or NFS is independently associated with colorectal space-occupying lesions， and liver fibrosis assessed by NFS might be associated with the increase in the number of lesions.

OBJECTIVE To investigate the effects of MIR149 rs2292832 and MIR499 rs3746444 gene polymorphisms on the efficacy of tofacitinib combined with iguratimod in the treatment of moderate‑to‑severe rheumatoid arthritis （RA）， to provide genetic reference for individualized treatment of moderate‑to‑severe RA patients.METHODS This was a prospective cohort study， and patients were grouped according to genotyping results. A total of 320 patients with moderate-to-severe RA diagnosed in the department of immunology of our hospital between January 2023 and January 2024 were enrolled. All patients received standardized treatment with tofacitinib （5 mg twice daily） combined with iguratimod （25 mg twice daily） for 24 weeks treatment course. Clinical efficacy outcomes， including American College of Rheumatology （ACR） 20/50/70 responses， disease activity score‑28 based on C‑reactive protein （DAS28-CRP）， erythrocyte sedimentation rate （ESR） and C-reactive protein （CRP）， were compared among patients with different genotypes. Combined-genotype analysis and multivariate Logistic regression analysis were also performed.RESULTS The MIR149 rs2292832 CC-genotype patients exhibited significantly higher ACR20， ACR50 and ACR70 response rates （88.90%， 67.78%， 45.56%） than those carrying CT- and TT-genotypes （ P ＜0.05）. After 24 weeks treatment， the reductions in DAS28‑CRP， ESR and CRP were significantly greater in CC‑genotype patients than in CT‑ and TT‑genotype patients （ P ＜0.05）， and the 24‑week DAS28‑CRP was significantly lower in the CC‑genotype patients than in CT‑ and TT‑genotype patients （ P ＜0.05）. The MIR499 rs3746444 GG-genotype showed significantly higher ACR20， ACR50 and ACR70 response rates （91.46%， 73.17%， 52.44%） than those carrying AG‑ and AA‑genotypes （ P ＜0.05）. After 24 weeks of treatment， the reductions in DAS28‑CRP， ESR and CRP were significantly greater in GG‑genotype patients than in AG- and AA-genotype patients and the 24‑weeks DAS28‑CRP was significantly lower in the GG‑genotype patients than in AG- and AA-genotype patients （ P ＜0.05）. Among the nine combined‑genotyps， the CC+GG genotype yielded the best treatment response. The MIR149 rs2292832 CC and MIR499 rs3746444 GG genotypes were independent predictors of clinical remission （ P ＜0.01）， whereas baseline DAS28‑CRP ≥ 6.5 served as a negative predictor of clinical remission （ P ＜0.05）.CONCLUSIONS Gene polymorphisms of MIR149 rs2292832 and MIR499 rs3746444 significantly influence the therapeutic effect of tofacitinib combined with iguratimod in patients with moderate-to-severe RA. Patients carrying CC and GG genotypes achieve better treatment responses.

Background: Influenza A viruses (IAVs) are the major pathogens associated with respiratory infections which can result in extensive pathological damage in lungs and serious complications. Isorhamnetin, an abundant natural flavonoid in fruits and medicinal plants, has recently been shown to have strong antioxidative, anti-inflammatory, and antiviral effects. Objective: This study investigated the pharmacological effects of isorhamnetin on viral pneumonia and explored the underlying mechanisms by in vivo and in vitro experiments. Materials and methods: In the present study, the protective effect of isorhamnetin against IAV was evaluated by the cytopathogenic effect assay, cell counting kit-8 assay, real-time polymerase chain reaction, and immunofluorescence assay in vitro. Then the pathological damage associated with pneumonia was examined by calculating the pulmonary index and performing micro-CT and hematoxylin-eosin staining in vivo. Thereafter, the related protein or gene levels of factors in the mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathways were determined by Western blot and immunofluorescence staining. Results: Isorhamnetin exerted significant anti-influenza effects and inhibited the expression of viral RNA in A549 cells, counteracting oxidative stress and apoptosis by suppressing the production of reactive oxygen species and caspase-3. The in vivo experiment results showed that isorhamnetin (20 and 40 mg/kg) caused a significant decrease in the pulmonary index, ameliorated pathological damage in the lung tissue, decreased viral load and NA activity, and reduced cytokines and nuclear factors. Furthermore, isorhamnetin could counteract the B cell lymphoma-2/B cell lymphoma-2–associated X protein (Bax) imbalance induced by PR8, suppress activation of the MAPK pathway, and upregulate the expression of Nrf2 and HO-1. Conclusions: Isorhamnetin can protect against viral pneumonia by activating the Nrf2/HO-1 pathway and suppressing the MAPK path-way. This study deciphers the pharmacological mechanism of isorhamnetin in alleviating pathological damage in viral pneumonia and provides rationale for the application of isorhamnetin in influenza treatment.

Background: Influenza A viruses (IAVs) are the major pathogens associated with respiratory infections which can result in extensive pathological damage in lungs and serious complications. Isorhamnetin, an abundant natural flavonoid in fruits and medicinal plants, has recently been shown to have strong antioxidative, anti-inflammatory, and antiviral effects. Objective: This study investigated the pharmacological effects of isorhamnetin on viral pneumonia and explored the underlying mechanisms by in vivo and in vitro experiments. Materials and methods: In the present study, the protective effect of isorhamnetin against IAV was evaluated by the cytopathogenic effect assay, cell counting kit-8 assay, real-time polymerase chain reaction, and immunofluorescence assay in vitro. Then the pathological damage associated with pneumonia was examined by calculating the pulmonary index and performing micro-CT and hematoxylin-eosin staining in vivo. Thereafter, the related protein or gene levels of factors in the mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathways were determined by Western blot and immunofluorescence staining. Results: Isorhamnetin exerted significant anti-influenza effects and inhibited the expression of viral RNA in A549 cells, counteracting oxidative stress and apoptosis by suppressing the production of reactive oxygen species and caspase-3. The in vivo experiment results showed that isorhamnetin (20 and 40 mg/kg) caused a significant decrease in the pulmonary index, ameliorated pathological damage in the lung tissue, decreased viral load and NA activity, and reduced cytokines and nuclear factors. Furthermore, isorhamnetin could counteract the B cell lymphoma-2/B cell lymphoma-2–associated X protein (Bax) imbalance induced by PR8, suppress activation of the MAPK pathway, and upregulate the expression of Nrf2 and HO-1. Conclusions: Isorhamnetin can protect against viral pneumonia by activating the Nrf2/HO-1 pathway and suppressing the MAPK path-way. This study deciphers the pharmacological mechanism of isorhamnetin in alleviating pathological damage in viral pneumonia and provides rationale for the application of isorhamnetin in influenza treatment.

Background: Influenza A viruses (IAVs) are the major pathogens associated with respiratory infections which can result in extensive pathological damage in lungs and serious complications. Isorhamnetin, an abundant natural flavonoid in fruits and medicinal plants, has recently been shown to have strong antioxidative, anti-inflammatory, and antiviral effects. Objective: This study investigated the pharmacological effects of isorhamnetin on viral pneumonia and explored the underlying mechanisms by in vivo and in vitro experiments. Materials and methods: In the present study, the protective effect of isorhamnetin against IAV was evaluated by the cytopathogenic effect assay, cell counting kit-8 assay, real-time polymerase chain reaction, and immunofluorescence assay in vitro. Then the pathological damage associated with pneumonia was examined by calculating the pulmonary index and performing micro-CT and hematoxylin-eosin staining in vivo. Thereafter, the related protein or gene levels of factors in the mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathways were determined by Western blot and immunofluorescence staining. Results: Isorhamnetin exerted significant anti-influenza effects and inhibited the expression of viral RNA in A549 cells, counteracting oxidative stress and apoptosis by suppressing the production of reactive oxygen species and caspase-3. The in vivo experiment results showed that isorhamnetin (20 and 40 mg/kg) caused a significant decrease in the pulmonary index, ameliorated pathological damage in the lung tissue, decreased viral load and NA activity, and reduced cytokines and nuclear factors. Furthermore, isorhamnetin could counteract the B cell lymphoma-2/B cell lymphoma-2–associated X protein (Bax) imbalance induced by PR8, suppress activation of the MAPK pathway, and upregulate the expression of Nrf2 and HO-1. Conclusions: Isorhamnetin can protect against viral pneumonia by activating the Nrf2/HO-1 pathway and suppressing the MAPK path-way. This study deciphers the pharmacological mechanism of isorhamnetin in alleviating pathological damage in viral pneumonia and provides rationale for the application of isorhamnetin in influenza treatment.

OBJECTIVE: To explore the pathogenic mechanism of a child with Waardenburg syndrome type 4C due to a c.661_664dup (p.P222Lfs*60) variant of SOX10 gene through in vitro experiments. METHODS: A child diagnosed at the Handan First Hospital was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples were collected from the child and his parents. Following extraction of genomic DNA, trio-whole exome sequencing was carried out. Pathogenicity of candidate variant was determined by bioinformatic analysis and reference to the guidelines from the American College of Medical Genetics and Genomics (ACMG). Candidate variant was verified by Sanger sequencing. Expression plasmids of wild-type SOX10 and the c.661_664dup (p.P222Lfs*60) variant were constructed and transiently transfected into 293T cells to determine the expression at the RNA and protein levels. The 293T cells transiently transfected with the wild-type/mutant SOX10 were treated with 10 ug/mL cycloheximide (CHX) for 0, 4, 8, 24 h, respectively, and the degradation rate of target protein was detected by Western blotting assay. This study has been approved by the Ethics Committee of Handan First Hospital (Ethics No. HDYY-LW-25053). RESULTS: The child was found to harbor a heterozygous c.661_664dup (p.P222Lfs*60) variant of the SOX10 gene, which was unreported previously. The variant did not significantly alter the expression of SOX10 at the mRNA level but the protein level. After the CHX treatment, the degradation of mutant SOX10 protein had slowed down. CONCLUSION The mutant SOX10 may affect the expression of downstream genes by affecting the degradation rate of its protein product.
Humans ; HEK293 Cells ; Mutation ; SOXE Transcription Factors/metabolism* ; Waardenburg Syndrome/genetics* ; Child

Objective: This study aims to investigate the therapeutic effects and underlying mechanisms of Xuanfei Baidu Decoction (XFBD) in a mouse model of dampness-heat toxin pneumonia. By exploring how XFBD exerts its effects, we seek to deepen our understanding of its role in treating pulmonary diseases and to address the current knowledge gap regarding its mechanisms of action, thereby supporting its clinical application. Methods: Ultra-high-performance liquid chromatography and high-resolution mass spectrometry (HRMS) were employed to analyze the chemical constituents of XFBD. The protective effects of XFBD were evaluated using a dampness-heat toxin-induced mouse model, established through dampness-heat exposure and HCoV-229E infection. XFBD was administered orally, followed by assessments including lung index measurement, micro-CT imaging, viral load quantification, cytokine analysis, and histological evaluation via hematoxylin-eosin staining. Proteomics and single-cell transcriptomic analyses were conducted to explore the potential mechanisms underlying XFBD’s pharmacological effects. A cellular model of HCoV-229E infection was developed to investigate changes in the cAMP/PKA signaling pathway. Molecular docking and surface plasmon resonance (SPR) experiments confirmed the strong binding affinity between key XFBD components and PKA. Finally, PKA activators and inhibitors were applied in vitro to validate these mechanistic findings. Results: In vivo studies demonstrated that XFBD significantly reduced the lung index, improved the structural integrity of lung and tongue tissues, and decreased levels of proinflammatory mediators, including IL-6, IL-8, and TNF-α. Proteomic and single-cell transcriptomic analyses showed that the differentially expressed proteins after XFBD treatment were primarily associated with inflammatory responses and immune regulation. The cAMP/PKA signaling pathway was identified as a key mechanism underlying these therapeutic effects. Notably, Western blot, ELISA, molecular docking, and SPR analyses confirmed that XFBD elevated cAMP levels and p-PKA expression, thereby activating the cAMP/PKA signaling pathway in vitro. Conclusion: This study demonstrated that XFBD significantly alleviates symptoms in mice with dampness-heat toxin pneumonia. Its therapeutic effects are mediated, at least in part, through activation of the cAMP/PKA signaling pathway. These findings provide compelling evidence that XFBD is an effective herbal remedy against HCoV-229E infection.

Objective To observe the effects of electroacupuncture on RhoA/ROCK2 signaling pathway in cerebral cortex of mice with amyotrophic lateral sclerosis(ALS);To explore the mechanism of electroacupuncture in improving the motor function of ALS mice.Methods The male hSOD1G93A mice were divided into model group and electroacupuncture group,and wild-type male mice in the same litter were set as blank group,with 12 mice in each group.After the mice were 60 days old,"Baihui"and both side of"Tianzhu","Tianshu"were selected for electroacupuncture for 10 min per day,5 days of continuous treatment and 2 days off for 3 weeks.Rotating rod experiment and open field experiment were used to evaluate the motor function of mice,the damage of neurons in cerebral cortex was observed by Nissl staining,Western blot was used to detect the expressions of Tar DNA binding protein-43(TDP-43),tumor necrosis factor(TNF)-α,interleukin(IL)-1β,RhoA and ROCK2 protein in cerebral cortex.Immunofluorescence staining was used to detect the positive expressions of ion calcium binding adapter molecule 1(Iba-1)and glial fibrillary acidic protein(GFAP)in cerebral cortex.Results Compared with the blank group,the incubation period of rod turning and the total distance of open field movement in the model group were reduced(P<0.01),the neuron damage was obvious in the cerebral cortex,with Nissl body shrinkage and reduction in quantity(P<0.01),the relative expressions of TDP-43,TNF-α,IL-1β,RhoA and ROCK2 protein increased(P<0.01),and the positive expression of Iba-1 and GFAP increased(P<0.01).Compared with the model group,the incubation period of rod turning and the total distance of open field movement increased in electroacupuncture group(P<0.05),the damage of neuron in cerebral cortex was reduced,the number of Nissl bodies increased(P<0.05),the expressions of TDP-43,TNF-α,IL-1β,RhoA and ROCK2 decreased(P<0.05),and the positive expression of Iba-1 and GFAP reduced(P<0.05).Conclusion Electroacupuncture can improve motor function in ALS model mice,and the mechanism may be related to the inhibition of RhoA/ROCK2 signaling pathway activity and then relieve neuroinflammation.

Objective To explore the characteristics and influencing factors of complicated intra-abdominal infection in ICU patients after abdominal surgery.Methods A retrospective study was performed on ICU patients(n=92,observation group)developing complicated intra-abdominal infection after abdominal surgery versus patients(n=104,control group)without complicated intra-abdominal infection after abdominal surgery in our hospital from January 2020 to December 2023.The characteristics of patients who developed complicated intra-abdominal infections were analyzed and the relevant influencing factors were identified using multivariate Logistic regression analysis.Results A total of 124 strains of pathogenic bacteria were isolated and identified in 92 infected patients,with 64.52％(80/124)of Gram-negative strains comprising mainly 29.03％(36/124)of Escherichia coli,25.81％(32/124)of Gram-positive strains comprising mainly 12.90％(16/124)of Enterococcus faecalis,and 9.68％(12/124)of fungi comprising mainly 6.45％(8/124)of Candida albicans.Multivariate Logistic regression results showed that preoperative underlying disease,surgical methods,duration of surgery,invasive procedures,antibiotic use,and length of ICU stay,enteral nutrition were the influencing factors of complicated intra-abdominal infection in ICU patients after abdominal surgery(P＜0.0 5).Conclusion Patients with complicated intra-abdominal infection are infected with a variety of pathogenic bacteria,predominantly Gram-negative.The patient's preoperative underlying disease,surgical approach,duration of surgery,invasive procedures,use of antibiotics,length of ICU stay,and early enteral nutrition all affect the risk of complicated intra-abdominal infection in ICU patients after abdominal surgery.