OBJECTIVE To investigate the improvement effects and mechanism of Achyranthes bidentata total saponins （ABS） extract on vascular endothelial dysfunction in spontaneously hypertensive rat （SHR） based on cytochrome P450 4A （CYP4A）/20-hydroxyeicosatetetraenoic acid （20-HETE）/G protein-coupled receptor 75 （GPR75） axis. METHODS Ten Wistar- Kyoto rats were taken as the normal control group. Forty SHR were first stratified by systolic blood pressure and then， within each stratum， randomly assigned using a random-number table to the model group （MOD group）， captopril positive control group （CAP group， 10 mg/kg）， ABS low- and high-dose extract groups （ABS-L group， ABS-H group， 60 and 120 mg/kg）， with 10 rats in each group. Animals in each group were given the corresponding drug or equal volume of pure water by gavage， once a day， for 28 consecutive days. After the last administration， systolic blood pressure of rats was measured. The levels of vasoactive substances， inflammatory factors and oxidative stress indicators in serum were measured. The pathological changes of rat thoracic aorta were observed. The level of reactive oxygen species （ROS） in aortic tissue was analyzed. The expressions of endothelial nitric oxide synthase （eNOS）， CYP4A， GPR75， nuclear factor-κB p65 （NF-κB p65）， phosphorylated NF-κB p65， p22phox， and reduced nicotinamide adenine dinucleotide phosphate oxidase 4（NOX4） in thoracic aorta tissue were detected. RESULTS After 28 d of treatment， compared with MOD group， the systolic blood pressure of rats in the ABS-L and ABS-H groups decreased significantly. The levels of 20-HETE， angiotensin Ⅱ， interleukin-1β， interleukin-6， tumor necrosis factor-α， intercellular cell adhesion molecule-1 and malondialdehyde in serum were significantly reduced （P＜0.05 or P＜0.01）， while the levels of nitric oxide， superoxide dismutase， glutathione peroxidase and catalase were significantly increased （P＜0.05 or P＜0.01）. Intimal damage of thoracic aorta was reduced， and endothelial cell morphology was improved. The expressions of ROS， CYP4A， GPR75， p22phox， NOX4 and the phosphorylation level of NF-κB p65 protein in thoracic aorta were down-regulated or reduced （P＜0.05 or P＜0.01）， while the expression of eNOS was up-regulated （P＜0.05 or P＜0.01）. CONCLUSIONS ABS extract may alleviate the inflammatory response and oxidative stress in SHR effectively by down-regulating the expression of CYP4A， reducing the production of 20-HETE， inhibiting the activation of GPR75， and subsequently suppressing the activation of downstream NF-κB and NOX4， thereby improving hypertension-related vascular endothelial dysfunction.

ObjectiveTo investigate the intervention effects of modified Linggui Zhugan Tang （LGZGT） on patients with obstructive sleep apnea-hypopnea syndrome （OSAHS） of the spleen deficiency and dampness obstruction with blood stasis type， reveal its possible mechanisms， and provide a theoretical basis for the clinical treatment of OSAHS with traditional Chinese medicine （TCM）. MethodsEighty OSAHS patients with spleen deficiency and dampness obstruction with blood stasis were randomly assigned to a control group and an observation group （1∶1） using a random number table， with 40 patients in each group. The control group received standard basic treatment combined with oral Doxofylline tablets， while the observation group received standard basic treatment combined with modified LGZGT. Serum levels of macrophage migration inhibitory factor （MIF）， microRNA-223 （miR-223）， and interleukin-18 （IL-18） were measured by enzyme-linked immunosorbent assay （ELISA）， and the mRNA expression levels of MIF， miR-223， and IL-18 were measured by real-time quantitative polymerase chain reaction （Real-time PCR）. After two months of treatment， the total clinical efficacy， apnea-hypopnea index （AHI）， lowest nocturnal oxygen saturation （LSpO₂）， body mass index （BMI）， TCM syndrome scores， and expression levels of MIF， miR-223， and IL-18 before and after treatment were compared between the two groups. Correlations between MIF， miR-223， IL-18 and AHI and LSpO₂ were also analyzed. ResultsCompared with the control group， the observation group showed a significantly higher total clinical effective rate （P<0.01， Z=-3.49）. Within the control group， no significant changes were observed in AHI， LSpO₂， BMI， TCM syndrome scores， or MIF， miR-223， IL-18 levels and their mRNAs after treatment. In the observation group， AHI， BMI， TCM syndrome scores， and MIF and IL-18 levels and their mRNAs decreased significantly， while LSpO₂ increased significantly （P<0.01）. After treatment， compared with the control group， the observation group exhibited significantly lower AHI， BMI， TCM syndrome scores， and MIF and IL-18 levels and their mRNAs， and significantly higher LSpO₂ （P<0.01）. Correlation analysis showed that MIF and IL-18 were positively correlated with AHI （P<0.01） and negatively correlated with LSpO₂ （P<0.01）， whereas miR-223 was negatively correlated with AHI （P<0.01） and positively correlated with LSpO₂ （P<0.01）. ConclusionModified LGZGT may improve OSAHS of the spleen deficiency and dampness obstruction with blood stasis type by reducing airway inflammatory factors， alleviating airway inflammation， relieving airway edema and stenosis， and improving airway obstruction.

Objective To investigate the predictive value of Mayo adhesion probability(MAP)score for intraoperative bleeding during microchannel percutaneous nephrolithotomy(PCNL), so as to provide reference for optimizing surgical decisions.Methods A retrospective analysis was conducted on the clinical data of 86 patients who underwent microchannel PCNL at our hospital during Jan.2018 and Jul. 2025.Patients were divided into two groups based on preoperative CT-derived MAP scores:MAP score＜3 group(n=45)and MAP score≥3 group(n=41).Baseline characteristics, surgical parameters and outcome measures were compared between the two groups.Univariate and multivariate logistic regression analyses were performed to identify risk factors for intraoperative bleeding(defined as a postoperative hemoglobin decrease of ≥20 g/L). Results The incidence of intraoperative bleeding was significantly higher in the MAP score≥3 group than in the MAP score＜3 group(43.9% vs.20.0%, P=0.017).Multivariate analysis identified MAP score ≥3(OR=3.045, 95%CI:1.125-9.547, P=0.019), use of anticoagulant medication(OR=3.016, 95%CI:1.251-9.257, P=0.020), establishment of multiple tracts(OR=5.445, 95%CI:1.798-17.578, P=0.003), and operation time ≥90 minutes(OR=5.697, 95%CI:1.890-13.632, P=0.001)as risk factors for intraoperative bleeding.The area under the curve for the MAP score in predicting intraoperative bleeding was 0.704.Conclusion The MAP score is an independent risk factor for intraoperative bleeding during microchannel PCNL.Preoperative assessment can help identify high-risk patients and provide a basis for optimizing surgical decision-making.

ObjectiveTo investigate the protective effect of Qiju Dihuang Wan on diabetic retinopathy （DR） and its regulatory mechanism on the NOD-like receptor protein 3/Cysteinyl aspartate-specific proteinase-1/Gasdermin D （NLRP3/Caspase-1/GSDMD） signaling pathway. MethodsHuman retinal microvascular endothelial cells （HRMEC） were divided into the blank group， model group， and Qiju Dihuang Wan low- and high-dose groups. Except for the blank group， all groups were induced with 40 mmol·L^-1 high glucose， and the treatment groups were supplemented with 6% and 12% Qiju Dihuang Wan-containing serum， respectively， based on the induction. After 24 h of culture， the levels of interleukin （IL）-1β and IL-18 were measured by enzyme-linked immunosorbent assay （ELISA）， pyroptosis-related protein expression was detected by immunofluorescence and Western blot， and the mRNA levels of NLRP3/Caspase-1/GSDMD signaling pathway-related molecules were determined by real-time polymerase chain reaction （Real-time PCR）. The mice were randomly divided into the blank group， model group， Qiju Dihuang Wan low-， medium-， and high-dose groups （11， 22， 44 g·kg^-1·d^-1）， and calcium dobesilate group （0.13 g·kg^-1·d^-1）. Except for the blank group， all groups were intraperitoneally injected with streptozotocin to establish the DR model. After successful modeling， the treatment groups received corresponding doses of Qiju Dihuang Wan crude drug or calcium dobesilate suspension by gavage once daily， while the blank group and model group received an equal volume of normal saline， for 8 consecutive weeks. Fundus changes were examined by optical coherence tomography， optical coherence tomography angiography， and fundus fluorescein angiography. Retinal pathological morphology was observed by hematoxylin-eosin staining. Real-time PCR was used to detect the mRNA levels of NLRP3， Caspase‑1， GSDMD， IL‑1β， and IL‑18 in retinal tissue， and Western blot was used to detect the protein expression of NLRP3， ASC， cleaved Caspase‑1， and GSDMD‑N. ResultsCell experiments showed that compared with the blank group， the model group showed significantly decreased HRMEC viability （P<0.01）， and significantly increased levels of IL-1β， IL-18， as well as increased protein expression of NLRP3， cleaved Caspase-1， and GSDMD-N （P<0.01）. Compared with the model group， the above indicators were significantly reduced in the high-dose Qiju Dihuang Wan groups （P<0.05， P<0.01）. Animal experiments showed that compared with the blank group， the model group had elevated blood glucose （P<0.01）， decreased retinal thickness and vascular density （P<0.01）， up‑regulated mRNA expression of NLRP3， Caspase‑1， GSDMD， IL‑1β and IL‑18 in retinal tissue （P<0.01）， and up‑regulated protein expression of NLRP3， ASC， cleaved Caspase‑1 and GSDMD‑N （P<0.01）. Compared with the model group， the Qiju Dihuang Wan medium- and high‑dose groups showed decreased blood glucose （P<0.01）， increased retinal thickness and vascular density （P<0.01）， alleviated retinal pathological damage， and down‑regulated mRNA expression of NLRP3， Caspase‑1， GSDMD， IL‑1β， IL‑18， as well as protein expression of NLRP3， ASC， cleaved Caspase‑1， and GSDMD‑N （P<0.05， P<0.01）. ConclusionQiju Dihuang Wan may exert protective effects against diabetic retinopathy by inhibiting the activation of the NLRP3/Caspase-1/GSDMD signaling pathway and alleviating high glucose-induced retinal pyroptosis.

ObjectiveTo investigate the protective effect of Qiju Dihuang Wan on diabetic retinopathy （DR） and its regulatory mechanism on the NOD-like receptor protein 3/Cysteinyl aspartate-specific proteinase-1/Gasdermin D （NLRP3/Caspase-1/GSDMD） signaling pathway. MethodsHuman retinal microvascular endothelial cells （HRMEC） were divided into the blank group， model group， and Qiju Dihuang Wan low- and high-dose groups. Except for the blank group， all groups were induced with 40 mmol·L^-1 high glucose， and the treatment groups were supplemented with 6% and 12% Qiju Dihuang Wan-containing serum， respectively， based on the induction. After 24 h of culture， the levels of interleukin （IL）-1β and IL-18 were measured by enzyme-linked immunosorbent assay （ELISA）， pyroptosis-related protein expression was detected by immunofluorescence and Western blot， and the mRNA levels of NLRP3/Caspase-1/GSDMD signaling pathway-related molecules were determined by real-time polymerase chain reaction （Real-time PCR）. The mice were randomly divided into the blank group， model group， Qiju Dihuang Wan low-， medium-， and high-dose groups （11， 22， 44 g·kg^-1·d^-1）， and calcium dobesilate group （0.13 g·kg^-1·d^-1）. Except for the blank group， all groups were intraperitoneally injected with streptozotocin to establish the DR model. After successful modeling， the treatment groups received corresponding doses of Qiju Dihuang Wan crude drug or calcium dobesilate suspension by gavage once daily， while the blank group and model group received an equal volume of normal saline， for 8 consecutive weeks. Fundus changes were examined by optical coherence tomography， optical coherence tomography angiography， and fundus fluorescein angiography. Retinal pathological morphology was observed by hematoxylin-eosin staining. Real-time PCR was used to detect the mRNA levels of NLRP3， Caspase‑1， GSDMD， IL‑1β， and IL‑18 in retinal tissue， and Western blot was used to detect the protein expression of NLRP3， ASC， cleaved Caspase‑1， and GSDMD‑N. ResultsCell experiments showed that compared with the blank group， the model group showed significantly decreased HRMEC viability （P<0.01）， and significantly increased levels of IL-1β， IL-18， as well as increased protein expression of NLRP3， cleaved Caspase-1， and GSDMD-N （P<0.01）. Compared with the model group， the above indicators were significantly reduced in the high-dose Qiju Dihuang Wan groups （P<0.05， P<0.01）. Animal experiments showed that compared with the blank group， the model group had elevated blood glucose （P<0.01）， decreased retinal thickness and vascular density （P<0.01）， up‑regulated mRNA expression of NLRP3， Caspase‑1， GSDMD， IL‑1β and IL‑18 in retinal tissue （P<0.01）， and up‑regulated protein expression of NLRP3， ASC， cleaved Caspase‑1 and GSDMD‑N （P<0.01）. Compared with the model group， the Qiju Dihuang Wan medium- and high‑dose groups showed decreased blood glucose （P<0.01）， increased retinal thickness and vascular density （P<0.01）， alleviated retinal pathological damage， and down‑regulated mRNA expression of NLRP3， Caspase‑1， GSDMD， IL‑1β， IL‑18， as well as protein expression of NLRP3， ASC， cleaved Caspase‑1， and GSDMD‑N （P<0.05， P<0.01）. ConclusionQiju Dihuang Wan may exert protective effects against diabetic retinopathy by inhibiting the activation of the NLRP3/Caspase-1/GSDMD signaling pathway and alleviating high glucose-induced retinal pyroptosis.

ObjectiveTo investigate the mechanism underlying the weight loss effect of electrothermal acupuncture （ETA） in diet-induced obese （DIO） model mice based on the hypoxia-inducible factor-1α （HIF-1α）/sphingosine-1-phosphate （S1P）sphingosine-1-phosphate receptor 3 （S1PR3） signaling pathway-mediated macrophage polarization. MethodsAn obesity mouse model was established by continuously feeding at a high-fat diet （HFD） for 8 weeks. Twenty successfully modeled obese mice were randomly divided into a model group and an electrothermal acupuncture （ETA） group with 10 mice per group， both of which continued to receive the HFD till week 12. Another 10 mice were assigned to the normal group and fed a standard chow diet continuously for 12 weeks. Starting from week 9 of the experiment， mice in the ETA group received electrothermal acupuncture stimulation at Zhongwan （CV12）， bilateral Tianshu （ST25）， and bilateral Zusanli （ST36） for 30 minutes per session， with the instrument current set at （40±0.5）mA and a constant temperature of （40±0.5）℃. Mice in the normal group and the model group were restrained in the same manner for 30 minutes without acupuncture. All interventions were performed once every other day for a total of 4 weeks. Ten hours after the final intervention， the body weight， waist circumference， and body length of mice in each group were measured， and Lee's index was calculated. Serum biochemical analysis was performed to assess fasting blood glucose （FBG）， total cholesterol （TC）， triglycerides （TG）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， blood urea nitrogen （BUN）， and creatinine （Cr）. Serum leptin （LEP） and adiponectin （ADPN） levels， as well as sphingosine-1-phosphate （S1P） levels in epididymal white adipose tissue （eWAT）， were detected by ELISA. Histomorphological changes in eWAT were monitored via HE staining. Flow cytometry was used to analyze macrophage subtype-related indexes in eWAT， including the total proportion of macrophages， the proportion of M1 macrophages， the proportion of M2 macrophages， and the M1/M2 ratio. Antibody array technology was used to determine the levels of differentially expressed inflammatory factors in eWAT between the model and blank groups， as well as between the ETA and model groups. Western blot and qPCR were used to detect the protein and mRNA expression levels of HIF-1α and S1PR3 in the eWAT， respectively. ResultsCompared to the normal group， the model group showed significantly increased body weight， Lee's index， waist circumference， and FBG， TC， TG， HDL-C， LDL-C， AST， and ALT levels， upgraded LEP and S1P levels， decreased ADPN level and ADPN/LEP ratio， elevated proportion of M1 macrophages and the M1/M2 ratio， reduced proportion of M2 macrophages， and upregulated protein and mRNA expression levels of HIF-1α and S1PR3 （P＜0.05 or P＜0.01）. With the exception of HDL-C， all the abovementioned indicators in the ETA group were significantly improved compared to the model group （P＜0.05 or P＜0.01）. Analysis of differentially expressed inflammatory cytokines revealed that， compared to the normal group， the levels of interleukin-1β （IL-1β）， interferon-γ （IFN-γ）， interleukin-6 （IL-6）， and interleukin-12p70 （IL-12p70） in the eWAT of the model group were elevated； conversely， the levels of interleukin-17 （IL-17）， interleukin-5 （IL-5）， IL-6， and interleukin-23 （IL-23） in the eWAT of the ETA group decreased compared to the model group （P＜0.05 or P＜0.01）. ConclusionETA exerts a weight-reducing effect in obese model mice， and the mechanism may involve inhibition of the HIF-1α/S1P/S1PR3 signaling pathway activation， which shifts macrophage polarization from the M1 to M2 phenotype， thereby alleviating chronic inflammation.

Objective To explore the role and mechanism of ferroptosis in vitamin D3(VD3)improving the cytotoxicity of keratinocytes(HaCaT cells)induced by nitrogen mustard(NM).Methods HaCaT cells were treated with gradient concentrations of NM(5,10,20,and 50 μmol/L)alone or in combination with ferroptosis inhibitor ferrostatin-1 or liproxstatin-1(10 μmol/L),Nrf2-specific agonist tBHQ(10 μmol/L),and VD3(10 nmol/L),respectively.After co-culture for 24 h,cell viability was assessed,and the intracellular contents of glutathione(GSH)and malondialdehyde(MDA)were measured.Additionally,the expression levels of Nrf2,xCT,and GPX4 were detected.Results NM exerted inhibitory effect on the proliferation of HaCaT cells in a dose-dependent manner,and the cell viability was reduced to approximately 55%at an NM concentration of 20 μmol/L(P<0.05).Ferroptosis inhibitors significantly mitigated the cytotoxic damage induced by NM in HaCaT cells(P<0.05),but activation of nuclear factor E2 related factor 2(Nrf2)markedly attenuated NM-induced ferroptosis,as indicated by the restoration of intracellular GSH level and the decrease in MDA content(P<0.05).VD3 specifically targeted the Nrf2 signaling pathway,upregulated the expression of xCT and GPX4 protein,thereby inhibiting ferroptosis and reducing NM-induced cytotoxicity in HaCaT cells.Conclusion VD3 mitigates NM-induced cytotoxicity in HaCaT cells by inhibiting ferroptosis via Nrf2 activation.

Objective To analyze the diversity and composition of gut microbiota in individuals with circadian rhythm disruption and their correlation with cognition.Methods Night shift workers and regular shift workers were subjected from our hospital during August 2022 and October 2024.The participants with circadian rhythm disorders were assigned into an experimental group(n=24),and those with normal circadian rhythms were into a control group(n=24).Their height,weight,age,gender,body mass index(BMI)and fresh fecal samples were collected,and Montreal Cognitive Assessment(MoCA)and Mini-Mental State Examination(MMSE)were used to evaluate their mental status.Metagenomics,Alpha and Beta diversity analyses,Linear Discriminant Analysis Effect Size(LEfSe),and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were employed to investigate the diversity and function characteristics of gut microbiota in the participants.Results There were no statistical differences between the 2 groups in baseline data,such as height,weight,gender,age,and BMI(P>0.05).Alpha diversity analysis indicated that no statistical differences were observed in the ACE,Chao1,Shannon,or Simpson indices between the 2 groups,while beta diversity analysis revealed significant differences(P<0.01),suggesting different structure of gut microbiota between them.In the experimental group,the abundance of Faecalibacterium prausnitzii and Agathobacter rectalis was decreased,while that of Escherichia coli and Phocaeicola vulgaratus was increased,with significant differences when compared with the control group(P<0.05).Additionally,KEGG functional analysis showed that the experimental group had obviously higher expression levels in Th17 cell differentiation and the IL-17 signaling pathway than the control group(P<0.05).Agathobacter rectalis and Faecalibacterium prausnitzii were positively correlated with MoCA score and MMSE score(P<0.05,P<0.01).Agathobacter rectalis was negatively correlated with the IL-17 signaling pathway and Th17 cell differentiation.Conclusion Individuals with circadian rhythm disorders have significant changes in the structure and function of gut microbiota when compared to those with normal circadian rhythms.Agathobacter rectalis may be involved in the regulation of the IL-17 signaling pathway and differentiation of Th17 cells,thereby possibly impacting the increases of cognitive score related to circadian rhythm disorders.

Objective To explore the effect of rotenone exposure on the metabolic homeostasis of nicotinamide adenine dinucleotide(NAD+)in dopaminergic neurons of the rat mid-brain striatum,and investigate the effect of exogenous NAD+intervention on the cellular damage response of dopaminergic neurons induced by rotenone.Methods Male SD rats(8 weeks old,200～250 g)were divided into a control group using a table of random numbers,a rotenone exposure group,an NAD+-intervention group,and an NAD+group.An intoxication model was established in the rotenone exposure group.NAD+(250 mg/kg)was administered simultaneously with rotenone exposure in the NAD+-intervention group.The NAD+group was only given NAD+,while the control group received no intervention.After modeling,open field test was performed to evaluate behavioral changes.After scarification,serum samples and mid-brain striatal tissues were collected.HE staining was used to observe the morphology of dopaminergic neurons in the striatum.The NAD+content in the tissues was detected with NAD+/NADH kit.Western blotting was employed to determine the contents of tyrosine hydroxylase(TH),nicotinamide phosphoribosyltransferase(NAMPT),nicotinamide mononucleotide adenylyltransferase(NMNAT),and solute carrier family 25 member A51(SLC25A51).ELISA was utilized to measure the content of dopamine in the striatal tissues.Immunohistochemical staining was applied to observe the distribution and contents of TH proteins in the striatal tissues of each group.Results Rotenone exposure significantly affected the vital signs and motor abilities of rats,induced disorderly-arranged,atrophy and deformed neurons in the striatal tissue,decreased the content of TH,rate-limiting enzyme for dopamine synthesis,by approximately 29%(P<0.01),the content of dopamine by about 42%,and that of NAD+by almost 50%(P<0.01),while increased the NADH/NAD+ratio(P<0.01).After exposure,the content of NAMPT,an enzyme related to NAD+synthesis,was decreased by 26%(P<0.05),the contents of NMNAT1-3 and SLC25A51,mitochondrial transporters of NAD+by approximately 21%,38%,43%,and 21%,respectively(P<0.01).Exogenous NAD+intervention improved the motor function of exposure rats and the morphology of dopaminergic neurons in the mid-brain striatal tissue,and restored the content of TH in the striatal tissue significantly by 12.8%(P<0.05),and the content of dopamine by 20.9%(P<0.05).Conclusion Rotenone disrupts the NAD+homeostasis in dopaminergic neurons by inhibiting the NAD+synthesis and transport pathways in the mid-brain striatal tissues,while exogenous NAD+intervention can effectively alleviate the dopaminergic neuron damage induced by rotenone exposure.

Objective To analyze the characteristics of methadone-related poisoning cases and provide a reference for forensic identification.Methods A total of 71 cases of methadone-related poisoning re-ported from 1998 to 2023 in China and 26 cases of methadone-related deaths reported from 2013 to 2018 in Italy were retrieved from databases including PubMed,Wanfang and CNKI.The general infor-mation,forensic pathological and toxicological characteristics were analyzed.Results Among the 71 methadone-related poisoning cases in China,there were 54 cases(76.06%)of poisoning without death and 17 cases(23.94%)of death from poisoning.There were 54 male cases(76.06%),and 51 cases(71.83%)aged 19 to 39 years old.There were 35 cases(49.30%)of poisoning caused by methadone alone,and 32 cases(45.07%)were poisoning caused by methadone combined with other substances or drugs including heroin and benzodiazepines.Most of the poisoned showed coma,respiratory depres-sion and miosis.Signs of asphyxia were often found by autopsy.The mass concentration of methadone detected in the blood of 6 deceased ranged from 0.112 to 3.000 mg/L.Among the 26 methadone-related deaths in Italy,22 cases were male(84.62%).There were 6 cases(23.08%)caused by methadone alone,and 20 cases(76.92%)died from methadone combined with other substances or drugs.The mass concentration of methadone in blood ranged from 0.181 to 4.059 mg/L.Conclusion The propor-tions of poisoning cases caused by methadone alone and methadone combined with other substances or drugs are comparable in China.The majority of deceased caused by methadone poisoning shows typi-cal triad of coma,respiratory depression and miosis,which helps forensic experts determine the cause of death related to methadone.Additionally,it is necessary to increase the routine testing of the con-centration of methadone and its combined substances or drugs in deceased,and collect data for the in-terpretation of the results of related cases.