Objective:To investigate the genetic mutation characteristics of patients with bilateral macronodular adrenocortical disease（BmacAD）and to analyze the clinical phenotypes and adrenal nodule transcriptomic profiles associated with different genotypes.Methods:This retrospective study analyzed the clinical data of 37 patients diagnosed with BmacAD by clinical and pathological assessment who underwent surgery at Peking Union Medical College Hospital from January 2001 to December 2024. Data included symptoms and signs related to hypercortisolism，24-hour urinary free cortisol，plasma adrenocorticotropic hormone（ACTH），and overnight low-dose dexamethasone suppression test results. Whole-exome sequencing（WES）was performed on frozen adrenal hyperplasia tissue samples from surgical specimens and matched frozen residual blood samples for all patients to screen for germline and somatic mutations. Sanger sequencing was used to validate some of the identified mutations. The relation between genetic mutations and clinical features was analyzed. Transcriptome sequencing of the hyperplastic tissues was conducted，and unsupervised clustering，differential gene expression analysis，and pathway enrichment analysis were used to compare the transcriptomic profiles of patients with different genotypes.Results:A total of 37 cases were included（23 males and 14 females），aged between 31?68 years old. Among the patients with available clinical data，most exhibited signs of hypercortisolism，including hypertension（100.0%，36/36），diabetes（50.0%，18/36），and centripetal obesity（55.9%，19/34），accompanied by relevant endocrine abnormalities such as elevated 24-hour urinary free cortisol（79.4%，27/34），suppressed ACTH（77.8%，28/36），and a positive overnight low-dose dexamethasone suppression test（45.2%，14/31）. WES and Sanger sequencing revealed that ARMC5 gene mutations were the most common. Among the 37 patients，33（89.2%）had ARMC5 germline mutations，and 31（83.8%）concurrently harbored ARMC5 somatic mutations，including 3 cases of loss of heterozygosity. Two patients without detectable ARMC5 mutations both carried a somatic p.S45P mutation in the CTNNB1 gene and neither had typical manifestations of hypercortisolism. Transcriptome sequencing results suggested that patients with ARMC5 mutations had an upregulation of pathways related to steroid secretion.Conclusions:The majority of BmacAD patients present with typical manifestations of hypercortisolism，while a minority exhibit only mild autonomous cortisol secretion without obvious symptoms. Biallelic inactivation of the ARMC5 gene is the primary genetic driver of this disease，leading to more severe hypercortisolism by upregulating steroid hormone secretion. CTNNB1 mutations may be involved in the pathogenesis of some patients without ARMC5 mutations.

Objective To evaluate the clinical efficacy of hip-knee-ankle active and passive exercise therapy in patients with early-to mid-stage knee osteoarthritis(KOA).Methods A total of 180 patients with early to mid-stage knee osteoarthritis(KOA)were recruited from the First Affiliated Hospital of Hebei University of Tradi-tional Chinese Medicine between March 2023 and March 2024.Patients were randomly assigned to one of four groups:active movement group,passive movement group,combined movement group,and control group,with 45 patients in each group.The active movement group received hip-knee-ankle active movement therapy daily until the end of follow-up.The passive movement group underwent hip-knee-ankle passive movement therapy three times per week for two weeks.The combined movement group received both active and passive therapies.The control group was administered oral celecoxib capsules(200 mg once daily for two weeks).Joint function was assessed in all four groups before treatment,at two weeks post-treatment,and at 14 weeks post-treatment.The primary outcome measure was the WOMAC joint function score,while secondary outcomes included the WOMAC pain score,stiffness score,and quality of life score(SF-12).Results A total of 160 patients completed the trial,with 39 in the active group,42 in the passive group,40 in the combined group,and 39 in the control group.There were no significant differences in baseline characteristics among the groups(P>0.05).Compared to baseline,the WOMAC scores for function,pain,and stiffness in the passive,combined,and control groups decreased significantly at both 2 and 14 weeks post-treatment(P<0.05),while the SF-12 scores increased significantly(P<0.05).Between 2 and 14 weeks post-treat-ment,the active and combined groups showed further significant decreases in WOMAC function,pain,and stiffness scores(P<0.05)and increases in SF-12 scores(P<0.05).At 2 weeks post-treatment,compared to the control group,the passive and combined groups exhibited significantly lower WOMAC function scores(P<0.05),with no significant difference between the passive and combined groups(P>0.05).By 14 weeks post-treatment,the active and combined groups demonstrated significantly lower WOMAC function scores(P<0.05),with the combined group showing a significantly lower score than the active group(P<0.05).Conclusion The four therapeutic approaches demonstrate a certain degree of efficacy in improving joint function for patients with early and mid-stage KOA.The passive therapy group exhibits superior short-term outcomes,while the active therapy group shows better long-term benefits.The combined therapy group presents notable advantages in both short-term and long-term effi-cacy,although its short-term effectiveness does not surpass that of the passive therapy group.It is recommended for patients with early and mid-stage KOA who have underlying gastrointestinal and cardiovascular conditions.

Objective To investigate the role of luteolin(Lut)in regulating reactive oxygen species(ROS)levels through nuclear factor erythroid 2-related factor 2(NFE2L2)/cystine glutamate antitransporter(x-CT)/glutathione peroxidase 4(GPX4)signaling axis to inhibit the viability of glioblastoma and promote apoptosis.Methods U87 MG and U251 cells were cultured in vitro.The CCK-8 assay was used to detect cell survival rates after 48 hours of treatment with different concentrations(0,6.25,12.5,25,50 and 100 μmol/L)of Lut.According to whether cells were treated with Lut,cells were divided into the U87 control group,the U87 Lut group,the U251 control group and the U251 Lut group.The half-maximal inhibitory concentration(IC50)at 48 hours was used as the unified treatment concentration for subsequent experiments.The apoptosis level of cells was detected by flow cytometry double staining method.Changes of reactive oxygen species(ROS)levels in cells were detected by the DCFH-DA method.Molecular docking was conducted using AutoDock software to verify the proteins related to the Lut and oxidative stress pathway.Real-time fluorescence quantitative reverse transcription(RT-qPCR)was used to detect the mRNA levels of NFE2L2 and GPX4.The expression levels of NFE2L2,x-CT and GPX4 proteins were detected by Western blot assay.Results After U87 MG and U251 cells were treated with Lut for 48 hours,the cell viability was significantly inhibited,and with the increase of Lut concentration,the cell viability decreased(P＜0.05).Compared with the U87 control group and the U251 control group respectively,the apoptosis rate of cells increased in the U87 Lut group and the U251 Lut group,the green fluorescence intensity was enhanced,and the intracellular ROS level was upregulated(P＜0.05).Results of molecular docking showed that Lut was tightly bound to NFE2L2,x-CT and GPX4.The results of RT-qPCR and Western blot assay showed that compared with the U87 control group and the U251 control group respectively,the protein and mRNA levels of NFE2L2 and GPX4 in cells of the U87 Lut group and the U251 Lut group,as well as the expression level of x-CT protein,decreased(P＜0.05).Conclusion Lut regulates ROS levels through the NFE2L2/x-CT/GPX4 signaling axis to inhibit the viability of glioblastoma and promote cell apoptosis.

Objective To evaluate the clinical efficacy of hip-knee-ankle active and passive exercise therapy in patients with early-to mid-stage knee osteoarthritis(KOA).Methods A total of 180 patients with early to mid-stage knee osteoarthritis(KOA)were recruited from the First Affiliated Hospital of Hebei University of Tradi-tional Chinese Medicine between March 2023 and March 2024.Patients were randomly assigned to one of four groups:active movement group,passive movement group,combined movement group,and control group,with 45 patients in each group.The active movement group received hip-knee-ankle active movement therapy daily until the end of follow-up.The passive movement group underwent hip-knee-ankle passive movement therapy three times per week for two weeks.The combined movement group received both active and passive therapies.The control group was administered oral celecoxib capsules(200 mg once daily for two weeks).Joint function was assessed in all four groups before treatment,at two weeks post-treatment,and at 14 weeks post-treatment.The primary outcome measure was the WOMAC joint function score,while secondary outcomes included the WOMAC pain score,stiffness score,and quality of life score(SF-12).Results A total of 160 patients completed the trial,with 39 in the active group,42 in the passive group,40 in the combined group,and 39 in the control group.There were no significant differences in baseline characteristics among the groups(P>0.05).Compared to baseline,the WOMAC scores for function,pain,and stiffness in the passive,combined,and control groups decreased significantly at both 2 and 14 weeks post-treatment(P<0.05),while the SF-12 scores increased significantly(P<0.05).Between 2 and 14 weeks post-treat-ment,the active and combined groups showed further significant decreases in WOMAC function,pain,and stiffness scores(P<0.05)and increases in SF-12 scores(P<0.05).At 2 weeks post-treatment,compared to the control group,the passive and combined groups exhibited significantly lower WOMAC function scores(P<0.05),with no significant difference between the passive and combined groups(P>0.05).By 14 weeks post-treatment,the active and combined groups demonstrated significantly lower WOMAC function scores(P<0.05),with the combined group showing a significantly lower score than the active group(P<0.05).Conclusion The four therapeutic approaches demonstrate a certain degree of efficacy in improving joint function for patients with early and mid-stage KOA.The passive therapy group exhibits superior short-term outcomes,while the active therapy group shows better long-term benefits.The combined therapy group presents notable advantages in both short-term and long-term effi-cacy,although its short-term effectiveness does not surpass that of the passive therapy group.It is recommended for patients with early and mid-stage KOA who have underlying gastrointestinal and cardiovascular conditions.

Objective To investigate the role of luteolin(Lut)in regulating reactive oxygen species(ROS)levels through nuclear factor erythroid 2-related factor 2(NFE2L2)/cystine glutamate antitransporter(x-CT)/glutathione peroxidase 4(GPX4)signaling axis to inhibit the viability of glioblastoma and promote apoptosis.Methods U87 MG and U251 cells were cultured in vitro.The CCK-8 assay was used to detect cell survival rates after 48 hours of treatment with different concentrations(0,6.25,12.5,25,50 and 100 μmol/L)of Lut.According to whether cells were treated with Lut,cells were divided into the U87 control group,the U87 Lut group,the U251 control group and the U251 Lut group.The half-maximal inhibitory concentration(IC50)at 48 hours was used as the unified treatment concentration for subsequent experiments.The apoptosis level of cells was detected by flow cytometry double staining method.Changes of reactive oxygen species(ROS)levels in cells were detected by the DCFH-DA method.Molecular docking was conducted using AutoDock software to verify the proteins related to the Lut and oxidative stress pathway.Real-time fluorescence quantitative reverse transcription(RT-qPCR)was used to detect the mRNA levels of NFE2L2 and GPX4.The expression levels of NFE2L2,x-CT and GPX4 proteins were detected by Western blot assay.Results After U87 MG and U251 cells were treated with Lut for 48 hours,the cell viability was significantly inhibited,and with the increase of Lut concentration,the cell viability decreased(P＜0.05).Compared with the U87 control group and the U251 control group respectively,the apoptosis rate of cells increased in the U87 Lut group and the U251 Lut group,the green fluorescence intensity was enhanced,and the intracellular ROS level was upregulated(P＜0.05).Results of molecular docking showed that Lut was tightly bound to NFE2L2,x-CT and GPX4.The results of RT-qPCR and Western blot assay showed that compared with the U87 control group and the U251 control group respectively,the protein and mRNA levels of NFE2L2 and GPX4 in cells of the U87 Lut group and the U251 Lut group,as well as the expression level of x-CT protein,decreased(P＜0.05).Conclusion Lut regulates ROS levels through the NFE2L2/x-CT/GPX4 signaling axis to inhibit the viability of glioblastoma and promote cell apoptosis.

Objective:To investigate the genetic mutation characteristics of patients with bilateral macronodular adrenocortical disease（BmacAD）and to analyze the clinical phenotypes and adrenal nodule transcriptomic profiles associated with different genotypes.Methods:This retrospective study analyzed the clinical data of 37 patients diagnosed with BmacAD by clinical and pathological assessment who underwent surgery at Peking Union Medical College Hospital from January 2001 to December 2024. Data included symptoms and signs related to hypercortisolism，24-hour urinary free cortisol，plasma adrenocorticotropic hormone（ACTH），and overnight low-dose dexamethasone suppression test results. Whole-exome sequencing（WES）was performed on frozen adrenal hyperplasia tissue samples from surgical specimens and matched frozen residual blood samples for all patients to screen for germline and somatic mutations. Sanger sequencing was used to validate some of the identified mutations. The relation between genetic mutations and clinical features was analyzed. Transcriptome sequencing of the hyperplastic tissues was conducted，and unsupervised clustering，differential gene expression analysis，and pathway enrichment analysis were used to compare the transcriptomic profiles of patients with different genotypes.Results:A total of 37 cases were included（23 males and 14 females），aged between 31?68 years old. Among the patients with available clinical data，most exhibited signs of hypercortisolism，including hypertension（100.0%，36/36），diabetes（50.0%，18/36），and centripetal obesity（55.9%，19/34），accompanied by relevant endocrine abnormalities such as elevated 24-hour urinary free cortisol（79.4%，27/34），suppressed ACTH（77.8%，28/36），and a positive overnight low-dose dexamethasone suppression test（45.2%，14/31）. WES and Sanger sequencing revealed that ARMC5 gene mutations were the most common. Among the 37 patients，33（89.2%）had ARMC5 germline mutations，and 31（83.8%）concurrently harbored ARMC5 somatic mutations，including 3 cases of loss of heterozygosity. Two patients without detectable ARMC5 mutations both carried a somatic p.S45P mutation in the CTNNB1 gene and neither had typical manifestations of hypercortisolism. Transcriptome sequencing results suggested that patients with ARMC5 mutations had an upregulation of pathways related to steroid secretion.Conclusions:The majority of BmacAD patients present with typical manifestations of hypercortisolism，while a minority exhibit only mild autonomous cortisol secretion without obvious symptoms. Biallelic inactivation of the ARMC5 gene is the primary genetic driver of this disease，leading to more severe hypercortisolism by upregulating steroid hormone secretion. CTNNB1 mutations may be involved in the pathogenesis of some patients without ARMC5 mutations.

Objective:To evaluate the effect of berberine on acute kidney injury (AKI) in rats undergoing liver transplantation and the role of AMP-activated protein kinase (AMPK).Methods:Twenty-four SPF-grade adult male Sprague-Dawley rats, aged 12 weeks, weighing 210-230 g, were divided into 4 groups ( n=6 each) using the random number table method: sham operation group (S group), AKI group, berberine group (BBR group), and berberine + AMPK inhibitor Compound C group (BBR-Comp C group). In BBR group, berberine 200 mg/kg was given by gavage starting from 2 weeks before surgery, once a day for 14 consecutive days. In BBR-Comp C group, Compound C 1 mg/kg was injected into the tail vein at 30 min before surgery. The rat AKI model was prepared by in situ liver transplantation in AKI group, BBR group and BBR-Comp C group. Blood specimens were taken from the inferior vena cava at 24 h postoperatively, and serum BUN and Cr concentrations were determined by enzyme-linked immunosorbent assay. Then the rats were sacrificed, and the kidney tissues were taken for microscopic examination of the pathological changes (with the light microscope after HE staining) and for determination of the expression of phosphorylated AMPK (p-AMPK), receptor-interacting protein kinase-1 (RIPK-1), receptor-interacting protein kinase-3 (RIPK-3) and mixed lineage kinase domain-like protein (MLKL) (by Western blot). Results:Compared with S group, the serum BUN and Cr concentrations were significantly increased, the p-AMPK expression was down-regulated, the expression of RIPK-1, RIPK-3 and MLKL was up-regulated ( P<0.05), and the pathological damage to renal tissues occurred in AKI group. Compared with AKI group, the serum BUN and Cr concentrations were significantly decreased, the p-AMPK expression was up-regulated, the expression of RIPK-1, RIPK-3 and MLKL was down-regulated ( P<0.05), and the pathological changes of renal tissues were significantly attenuated in BBR group. Compared with BBR group, the serum BUN and Cr concentrations were significantly increased, the p-AMPK expression was down-regulated, and the expression of RIPK-1, RIPK-3 and MLKL was up-regulated in BBR-Comp C group ( P<0.05). Conclusions:Berberine can attenuate AKI in rats undergoing liver transplantation, and the mechanism may be related to the promotion of AMPK phosphorylation and inhibition of programmed necrosis.

Objective To observe the clinical effect of traditional Chinese medicine bone-setting technique using spinal,pelvi-lower extremity line to treat patients with knee osteoarthritis(KOA).Methods 426 patients with KOA were all from the First Affiliated Hospital of Hebei University of Traditional Chinese Medicine.They were randomly divided into experimental group(384 cases,57 cases of elimination,shedding and termination)by computer generated sequence.Traditional Chinese bone setting techniques were applied with spinal-pelvic-lower limb force line(divided into three parts:lumbar fixed point reduction method,hip joint push-pull and extension method and knee peripheral tendon recovery method every 3 days.2 weeks)treatment;The control group was the waiting treatment group(48 cases,6 cases were eliminated,abscission,termination),which was only used for clinical observation for 2 weeks.The main outcome index was WOMAC pain score.Secondary outcome measures were WOMAC stiffness score,functional score,standardized score and quality of life score(SF-12).The test time points were baseline,2 weeks after enrollment,and follow-up(14 weeks after enrollment).The control group was at baseline and 2 weeks after enrollment.Results Compared with baseline,WOMAC pain score,stiffness score,functional score and standardized score were all decreased in 2 groups 2 weeks after enrollment(P<0.05),but the experimental group was significantly decreased compared with the control group(P<0.001).SF-12 quality of life scores were all higher than before(P<0.001),but the experimental group was significantly higher than the control group(P<0.001).At follow-up,compared with 2 weeks after enrollment,WOMAC pain scores were increased(P<0.001),WOMAC stiffness,joint function and standardized scores were decreased(P<0.001),and SF-12 scores were increased(P<0.001).Conclusion The use of spinal-pelvi-lower extremity line of traditional Chinese medicine bone-setting technique in the treatment of KOA is effective in improving the knee joint function and improving the quality of life of patients,but the short-term effect of pain relief is good,and the long-term effect is not good.Its safety is good,and it can be considered in clinical application for KOA patients with joint dysfunction as the main manifestation.

L-dopa (l-3,4-dihydroxyphenylalanine)-induced dyskinesia (LID) is a debilitating complication of dopamine replacement therapy for Parkinson's disease. The potential contribution of striatal D₂ receptor (D₂R)-positive neurons and downstream circuits in the pathophysiology of LID remains unclear. In this study, we investigated the role of striatal D₂R⁺ neurons and downstream globus pallidus externa (GPe) neurons in a rat model of LID. Intrastriatal administration of raclopride, a D₂R antagonist, significantly inhibited dyskinetic behavior, while intrastriatal administration of pramipexole, a D₂-like receptor agonist, yielded aggravation of dyskinesia in LID rats. Fiber photometry revealed the overinhibition of striatal D₂R⁺ neurons and hyperactivity of downstream GPe neurons during the dyskinetic phase of LID rats. In contrast, the striatal D₂R⁺ neurons showed intermittent synchronized overactivity in the decay phase of dyskinesia. Consistent with the above findings, optogenetic activation of striatal D₂R⁺ neurons or their projections in the GPe was adequate to suppress most of the dyskinetic behaviors of LID rats. Our data demonstrate that the aberrant activity of striatal D₂R⁺ neurons and downstream GPe neurons is a decisive mechanism mediating dyskinetic symptoms in LID rats.
Rats ; Animals ; Levodopa/toxicity* ; Dopamine ; Parkinsonian Disorders/drug therapy* ; Oxidopamine ; Dyskinesia, Drug-Induced ; Corpus Striatum/metabolism* ; Neurons/metabolism* ; Receptors, Dopamine D2/metabolism* ; Antiparkinson Agents/toxicity*

Objective:To evaluate the role of ferroptosis in lung injury in a rat model of autologous orthotopic liver transplantation.Methods:Twenty-four healthy adult SPF-grade male rats, aged 8-10 weeks, weighing 230-270 g, were divided into 3 groups ( n=8 each) using the random number table method: sham operation group (S group), autologous in situ liver transplantation group (LT group) and ferroptosis inhibitor Ferrostain-1 group (LT+ Fer-1 group). In LT group and LT+ Fer-1 group, an autologous in situ liver transplantation model was developed in anesthetized animals, and Ferrostain-1 5 mg/kg was intraperitoneally injected at 30 min before surgery in LT+ Fer-1 group. The inferior vena cava blood samples were obtained at 6 h of reperfusion, then animals were sacrificed, and lung tissues were obtained. The morphology of lung tissues was examined, and the lung injury was scored. The serum malondialdehyde (MDA) concentration and contents of MDA, reduced glutathione (GSH), glutathione peroxidase4 (GPX4), and Fe 2+ in lung tissues were measured by enzyme-linked immunosorbent assay. The expression of ferritin heavy chain 1 (FTH1) and solute carrier family 7 member 11 recombinant protein (SLC7A11) was determined by Western blot. Results:Compared with S group, the lung injury, serum MDA concentration, and contents of MDA and Fe 2+ were significantly increased, the contents of GSH and GPX4 were decreased, and the expression of FTH1 and SLC7A11 was down-regulated in LT group ( P<0.05). Compared with LT group, the lung injury, serum MDA concentration, and contents of MDA and Fe 2+ were significantly decreased, the contents of GSH and GPX4 were increased, and the expression of FTH1 and SLC7A11 was up-regulated in LT+ Fer-1 group ( P< 0.05). Conclusions:Ferroptosis is involved in the pathophysiology of lung injury in a rat model of autologous orthotopic liver transplantation.