BackgroundBoth modified Suanzaoren Decoction and estazolam serve as effective treatments for chronic insomnia disorder （CID）. Default mode network （DMN） dysfunction is recognized as the primary pathophysiological mechanism underlying CID. However， it remains unclear whether the two treatments exert their therapeutic effects via convergent modulation of DMN functional connectivity. ObjectiveTo investigate the convergent remodeling characteristics of bidirectional effective connectivity （EC） of core DMN nodes with both the intra-network and the remaining whole-brain regions in CID patients with Yin-deficiency and fire-hyperactivity syndrome following the treatment with modified Suanzaoren Decoction or estazolam， so as to provide neuroimaging evidence for the convergent neural mechanisms underlying the therapeutic effects of both therapies in treating CID. MethodsEighty-two patients diagnosed with CID according to the International Classification of Sleep Disorder， third edition （ICSD-3） were consecutively recruited from the outpatient clinic of the Inner Mongolia Autonomous Region Mental Health Center from October 2020 to September 2023. Based on treatment preference， patients were assigned to receive a 6-week intervention in either the modified Suanzaoren Decoction group （n=52） or the estazolam group （n=30）. Ultimately， 66 patients completed follow-up assessments （modified Suanzaoren Decoction group， n=41； estazolam group， n=25）. All CID patients underwent resting-state functional magnetic resonance imaging scanning before and after treatment. Core DMN nodes including medial prefrontal cortex （mPFC）， bilateral inferior parietal lobule （IPL）， and precuneus （PCUN） were selected as seed regions. Granger causality analysis （GCA） was employed to assess bidirectional EC between the seed regions and both the other intra-DMN nodes and the remaining whole-brain regions. Hepatic and renal function indices were detected pre- and post-treatment to evaluate the safety profiles of both treatments. ResultsAt baseline， significant between-group differences were identified in EC from the left IPL to the left middle fronto-orbital gyrus/gyrus rectus （t=5.24， Z=4.84， voxel-level P<0.001， cluster-level P<0.05， GRF-corrected）， with the modified Suanzaoren Decoction group exhibiting stronger EC than the estazolam group. No significant between-group differences were observed in EC between other core DMN nodes and remaining whole-brain regions （P>0.05）. Within-group pre- to post-treatment comparisons demonstrated consistent connectivity alterations in both groups. Specifically， EC from the left IPL to the supplementary motor area （SMA） decreased after treatment （modified Suanzaoren Decoction group： t=-6.28， Z=5.208； estazolam group： t=-5.58， Z=4.425）. Conversely， EC from the SMA to the left IPL increased （modified Suanzaoren Decoction group： t=5.89， Z=4.968； estazolam group： t=6.15， Z=4.720）. Furthermore， both groups demonstrated reduced EC from the PCUN to the left IPL （modified Suanzaoren Decoction group： t=-5.85， Z=4.94； estazolam group： t=-5.75， Z=4.515）， and elevated EC from the left IPL to the PCUN （modified Suanzaoren Decoction group： t=5.29， Z=4.579； estazolam group： t=4.55， Z=3.826） （voxel-level P<0.001， cluster-level P<0.05， GRF-corrected）. No significant pre- to post-treatment differences were observed in hepatic or renal function indices in either group （P>0.05）. ConclusionIn CID patients with Yin-deficiency and fire-hyperactivity syndrome， both modified Suanzaoren Decoction and estazolam induce convergent bidirectional remodeling of EC between the left IPL and the SMA and the PCUN. ［Funded by Natural Science Fund Project of Inner Mongolia Autonomous Region （number， 2019MS08099）； ClinicalTrials.gov number， NCT06452953］

AIM:To explore the central mechanisms by which metformin(Met)attenuates insulin resistance in high-fat diet(HF)-fed rats.METHODS:Forty healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal chow(NC)group,HF group,HF+Met group,and HF+Met+SHU9119[melanocortin 4 receptor(MC4R)antagonist]group,with 10 rats per group.Treatments with HF and Met lasted for 12 weeks,while SHU9119 was injected for the last 10 d.Skeletal muscle AMP-activated protein kinase(AMPK)and silent information regulator 1(SIRT1)ex-pression and activity were measured,along with mitochondria oxidative stress markers,mitochondrial function and quanti-ty.Systemic and skeletal muscle insulin sensitivity were assessed using the average glucose infusion rate from 60 to 120 min(GIR60-120)and 2-deoxyglucose uptake(DGU)during hyperinsulinemic-euglycemic clamp.RESULTS:The rats in HF group exhibited significantly reduced expression and activity of AMPK/SIRT1 in skeletal muscles(P<0.05).More-over,mitochondrial oxidative stress markers,reactive oxygen species(ROS)and malondialdehyde(MDA),were marked-ly elevated(P<0.05),and the activity of antioxidant enzymes glutathione peroxidase(GPX)and manganese superoxide dismutase(MnSOD)was significantly decreased in HF group(P<0.05).There was also a notable decline in the activity of citrate synthase(P<0.05),a marker of mitochondrial oxidative capacity,and the copy number of mitochondrial DNA in HF group.These changes were correlated with significantly decreased GIR60-120 and DGU(P<0.05).Notably,Met treat-ment(HF+Met)restored the AMPK/SIRT1 expression and activity,improved mitochondrial function,and reduced oxida-tive stress,leading to improved insulin sensitivity(P<0.05).However,these beneficial effects of Met were reversed by the MC4R antagonist SHU9119 in HF+Met+SHU9119 group.CONCLUSION:Treatment with Met enhances skeletal muscle AMPK/SIRT1 expression and activity,reverses mitochondrial dysfunction,and improves insulin resistance in HF-fed rats.These effects might be mediated through the activation of hypothalamic MC4R.

Objective To explore the effect of exenatide on oxidative stress in the hypothalamus of diabetes mice and its potential mechanism.Methods After one week of adaptive feeding,C57BL/6J mice were randomly divided into the CON group(normal chaw diet),the T2DM group(high-fat diet,HFD),and the T2DM+Exe group(HFD+exenatide).After 8 weeks of HFD,mice in the T2DM+Exe group were intraperitoneally injected with exenatide[24 nmol/(kg·d)]for 8 weeks.The weight and glucose and lipid metabolism levels of the mice were measured,and the levels of inflammatory and adipokine factors in mice were detected using the ELISA method.Western Blot was used to detect the expression of melanocortin receptor-4(MC4R)and proopiomelanocor-tin(POMC)in the hypothalamus.Hypothalamic mitochondria were extracted,and the content of mitochondrial reactive oxygen species(ROS)was measured using a flow cytometer.The content of malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)in the mitochondria were detected using assay kits.Changes in the ultrastructure of mitochondria were observed using a transmission electron microscope.In vitro experiments,pal-mitic acid(PA)and exenatide were used to treat hypothalamic GT1-7 cells,and short hairpin RNA(shRNA)was used to silence the melanocortin 4 receptor(MC4R),and observe the cellular oxidative stress and lipid deposition.Results Compared with the CON group,the T2DM group mice showed a significant increase in glucose and lipid metabolism indicators,pro-inflammatory factors,and adipose factor levels(P<0.05),the expression of MC4R and POMC proteins in the hypothalamus were decreased(P<0.05),and the mitochondrial ROS and MDA content in the hypothalamus significantly were increased(P<0.05),while SOD and CAT activities were decreased(P<0.05).Mitochondrial morphology was abnormal.After intervention with exenatide,the above indicators were signifi-cantly improved.After inhibiting MC4R expression in vitro experiments,compared with the intervention group with exenatide,the ROS and MDA content was significantly increased(P<0.05),SOD activity was decreased(P<0.05),and lipid deposition occurred in the cells.Conclusions Exenatide exhibits a protective effect on hypotha-lamic oxidative stress injury in diabetic mice,and this mechanism may be associated with the upregulation of MC4R expression.

AIM:To explore the central mechanisms by which metformin(Met)attenuates insulin resistance in high-fat diet(HF)-fed rats.METHODS:Forty healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal chow(NC)group,HF group,HF+Met group,and HF+Met+SHU9119[melanocortin 4 receptor(MC4R)antagonist]group,with 10 rats per group.Treatments with HF and Met lasted for 12 weeks,while SHU9119 was injected for the last 10 d.Skeletal muscle AMP-activated protein kinase(AMPK)and silent information regulator 1(SIRT1)ex-pression and activity were measured,along with mitochondria oxidative stress markers,mitochondrial function and quanti-ty.Systemic and skeletal muscle insulin sensitivity were assessed using the average glucose infusion rate from 60 to 120 min(GIR60-120)and 2-deoxyglucose uptake(DGU)during hyperinsulinemic-euglycemic clamp.RESULTS:The rats in HF group exhibited significantly reduced expression and activity of AMPK/SIRT1 in skeletal muscles(P<0.05).More-over,mitochondrial oxidative stress markers,reactive oxygen species(ROS)and malondialdehyde(MDA),were marked-ly elevated(P<0.05),and the activity of antioxidant enzymes glutathione peroxidase(GPX)and manganese superoxide dismutase(MnSOD)was significantly decreased in HF group(P<0.05).There was also a notable decline in the activity of citrate synthase(P<0.05),a marker of mitochondrial oxidative capacity,and the copy number of mitochondrial DNA in HF group.These changes were correlated with significantly decreased GIR60-120 and DGU(P<0.05).Notably,Met treat-ment(HF+Met)restored the AMPK/SIRT1 expression and activity,improved mitochondrial function,and reduced oxida-tive stress,leading to improved insulin sensitivity(P<0.05).However,these beneficial effects of Met were reversed by the MC4R antagonist SHU9119 in HF+Met+SHU9119 group.CONCLUSION:Treatment with Met enhances skeletal muscle AMPK/SIRT1 expression and activity,reverses mitochondrial dysfunction,and improves insulin resistance in HF-fed rats.These effects might be mediated through the activation of hypothalamic MC4R.

Objective To explore the effect of exenatide on oxidative stress in the hypothalamus of diabetes mice and its potential mechanism.Methods After one week of adaptive feeding,C57BL/6J mice were randomly divided into the CON group(normal chaw diet),the T2DM group(high-fat diet,HFD),and the T2DM+Exe group(HFD+exenatide).After 8 weeks of HFD,mice in the T2DM+Exe group were intraperitoneally injected with exenatide[24 nmol/(kg·d)]for 8 weeks.The weight and glucose and lipid metabolism levels of the mice were measured,and the levels of inflammatory and adipokine factors in mice were detected using the ELISA method.Western Blot was used to detect the expression of melanocortin receptor-4(MC4R)and proopiomelanocor-tin(POMC)in the hypothalamus.Hypothalamic mitochondria were extracted,and the content of mitochondrial reactive oxygen species(ROS)was measured using a flow cytometer.The content of malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)in the mitochondria were detected using assay kits.Changes in the ultrastructure of mitochondria were observed using a transmission electron microscope.In vitro experiments,pal-mitic acid(PA)and exenatide were used to treat hypothalamic GT1-7 cells,and short hairpin RNA(shRNA)was used to silence the melanocortin 4 receptor(MC4R),and observe the cellular oxidative stress and lipid deposition.Results Compared with the CON group,the T2DM group mice showed a significant increase in glucose and lipid metabolism indicators,pro-inflammatory factors,and adipose factor levels(P<0.05),the expression of MC4R and POMC proteins in the hypothalamus were decreased(P<0.05),and the mitochondrial ROS and MDA content in the hypothalamus significantly were increased(P<0.05),while SOD and CAT activities were decreased(P<0.05).Mitochondrial morphology was abnormal.After intervention with exenatide,the above indicators were signifi-cantly improved.After inhibiting MC4R expression in vitro experiments,compared with the intervention group with exenatide,the ROS and MDA content was significantly increased(P<0.05),SOD activity was decreased(P<0.05),and lipid deposition occurred in the cells.Conclusions Exenatide exhibits a protective effect on hypotha-lamic oxidative stress injury in diabetic mice,and this mechanism may be associated with the upregulation of MC4R expression.

Objective:To explore the optimal index of rotational displacement of femoral neck fractures by modeling the axial rotational displacement of femoral neck fractures after reduction and based on X-ray projections.Methods:Six dry human femur specimens, comprising 2 males and 4 females, were utilized in the study. Design and manufacture a proximal femur ortholateral and oblique X-ray casting jigs and mounts. The femoral neck fracture was modeled on the femoral specimen, with Pauwells 30°, 50°, and 70° models (2 each) made according to Pauwells typing. The fractures were manually repositioned with residual anterior 20°, 40° and 60° axial rotational displacements. Each fracture model was projected at different angles (pedicled 40°, pedicled 20°, vertical 0°, cephalad 20°, and cephalad 40°), and the trabecular angle and Garden's alignment index of the model were measured to observe the imaging characteristics of the fracture line on the medial oblique and lateral oblique radiographs.Results:In the presence of a 20° and 40° anterior rotational displacement following reduction of a femoral neck fracture, the trabecular angle in the rotationally displaced group was not significantly different from that of the anatomically repositioned group in various projection positions. However, when a residual rotational displacement of 60° was present, the trabeculae appeared blurred at most projection angles in the Pauwells 30° and 50° models, failing to measure trabecular angles. In the Pauwells 70° fracture model, the trabecular angle in the rotational displacement group was significantly different from that in the anatomical reduction group. In anteroposterior radiographs, when the anterior rotation displacement was 60° in the Pauwells 70° group, Garden's contralateral index showed an unsatisfactory restoration (150°, 142°), whereas all rotationally displaced models in the Pauwells 30° and Pauwells 50° groups had a Garden's contralateral index of >155°, which achieved an acceptable restoration. In lateral radiographs, all rotational displacement models with Garden's alignment index>180° failed to achieve acceptable repositioning, and the larger the Pauwells angle the greater the Garden's alignment index at the same rotational displacement. In the internal oblique position with a bias towards the foot side, the image showed partial overlap between the femoral head and the shaft, making it difficult to assess the quality of the reduction. Conversely, when projected cephalad, the femoral neck appeared longer, particularly at a projection angle of 40° cephalad, allowing for clear observation of the fracture line and the anatomy of the proximal femur. The trabeculae were not well visualized in the external oblique position.Conclusion:There are limitations in applying the trabecular angle to assess the axial rotational displacement of the femoral head after reduction of femoral neck fractures. The Pauwells 70° with residual rotational anterior displacement of 60° was the only way to detect axial rotational displacement of the femoral head on anteroposterior radiographs Garden's alignment index. For the determination of axial rotational displacement of the femoral head, the Garden's alignment index on lateral radiographs provides higher reliability.

Examining mechanisms involved in the mutual regulation between the muscular system and the skeletal system, elucidating the key issues responsible for loss of muscle and bone mass and strength, and thus halting the progression of these conditions are critical measures for reducing fractures caused by falls and subsequent disability and mortality.At present, most studies have treated the muscular system and the skeletal system separately, often ignoring the mutual regulation and connections between them.This article reviews the current research progress on the mechanisms of interaction between the two systems, aiming to provide a basis for the prevention, diagnosis and treatment of disuse-related diseases in the elderly population.

Humans ; Gastrointestinal Microbiome ; Constipation/therapy* ; Probiotics/therapeutic use* ; Patients

Objective　To evaluate the sensitivity and specificity of the GenoType MTBDRplus V2.0 (Mycobacterium tuberculosis and resistance gene detection assay kit using PCR-linear probe hybridization with enzyme chromogenic method, referred to as MTBDRplus 2.0) kit for detection of rifampin and isoniazid resistance, providing the basis for improving the detection of drug-resistant Mycobacterium tuberculosis. Methods From January to December 2022, positive strains of Mycobacterium tuberculosis isolated and cultured from designated tuberculosis treatment hospitals in 32 counties (cities, districts) of Yunnan Province were collected. Resistance in 880 strains of Mycobacterium tuberculosis was detected by MTBDRplus 2.0, and the minimum inhibitory concentration (MIC) method was used for the drug sensitivity test. 　　　Results Using the MIC method as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of MTBDRplus 2.0 in detecting isoniazid resistance were 67.69%, 98.40%, 77.19%, and 97.45%, respectively. The consistency of the linear probe method and MIC method in detecting isoniazid resistance was moderate, with a Kappa value of 0.701 (P<0.001); the sensitivity, specificity, positive predictive value, and negative predictive values of MTBDRplus 2.0 in detecting rifampicin resistance were 87.80%, 99.40%, 87.80%, and 99.40%, respectively. The consistency of rifampicin resistance detection between MTBDRplus 2.0 and MIC method was relatively good, with a Kappa value of 0.872 (P<0.001). Among the 13 strains showing resistance to isoniazid with MTBDRplus 2.0, but sensitivity according to the MIC method, 11 strains (84.62%) had mutations in the inhA gene (C15T). Out of the 5 strains showing resistance to rifampicin with MTBDRplus 2.0, but sensitivity according to the MIC method, 4 strains (80.00%) had other mutations in the rpoB gene. Conclusions MTBDRplus 2.0 shows high sensitivity and specificity in detecting rifampicin resistance, but slightly low sensitivity in detecting isoniazid resistance. The low sensitivity in detecting isoniazid resistance may be due to insufficient target coverage of the detection kit. inhA gene mutations are poorly correlated with isoniazid resistance, and other mutations in the rpoB gene are poorly correlated with rifampicin resistance.

OBJECTIVE To develop a high-throughput screening assay for the discovery of Omicron variant main protease(OM-Mpro) inhibitors based on the principle of fluorescence resonance energy transfer(FRET). METHODS The recombinant OM-Mpro enzyme was expressed in Escherichia coli Rosetta(DE3) cells, and further purified by a HisTrapTM chelating column. Subsequently, the enzymatic activity of OM-Mpro and wild type main protease(WT-Mpro) enzymes and inhibition of nirmatrelvir against both proteases were measured using FERT assay. With the FRET assay, OM-Mpro inhibitors were identified via high-throughput screening of an approved drug library. RESULTS The active OM-Mpro enzyme was successfully prepared from E. coli cells. OM-Mpro and WT-Mpro enzymes possessed the same enzymatic activity, and OM-Mpro remained susceptible to nirmatrelvir in vitro. Through high-throughput screening of the marketed drug library, it was found that cetylpyridinium chloride(CPC) is a mixed-type OM-Mpro inhibitor in vitro with an IC50 value of 8.76 μmol·L−1. CONCLUSION A robust FRET assay has been successfully developed based on the production of active OM-Mpro enzyme for screening of its inhibitors, and CPC is identified as a potential lead compound against OM-Mpro in vitro. This study provides a promising avenue for rapid discovery of broad-spectrum antivirals against coronavirus protease.