Objective:To analyze the infection status of Coxsackievirus B group (CVB) in regions affected by sudden unexplained death in Yunnan (referred to as sudden death in Yunnan), and to provide a scientific basis for formulating effective prevention and control strategies.Methods:A cross-sectional survey method was employed. The population from 16 counties (cities, districts, referred to as counties) affected by sudden death in Yunnan Province from 2002 to 2022 and the population from one non-affected county in 2021 and 2022 (control population) were classified into cases of sudden death in Yunnan (7 cases), co-occurring cases (29 cases), high-risk population (1 303 cases), and control population (270 cases). Blood samples were collected from these populations. By using enzyme-linked immunosorbent assay (ELISA), CVB immunoglobulin M (CVB-IgM) antibodies in the acute-phase serum samples of the population in the affected areas were detected, and CVB immunoglobulin G (CVB-IgG) antibodies in the convalescent-phase serum samples were detected. Both types of detections were carried out on the control population, and the test results were analyzed.Results:A total of 1 609 serum samples were tested, including 1 339 samples from the population in the affected areas (923 acute-phase samples and 416 convalescent-phase samples) and 270 samples from the control population. Among the 16 affected counties, positive CVB-IgM antibody results were detected in 9 counties. The overall positive rate of the population in the affected areas was higher than that of the control population [7.80% (72/923) vs. 4.44% (12/270), χ 2 = 40.78, P < 0.001]. The positive rates of the high-risk population in Dayao County and Lufeng City were both higher than that of the control population [(22.22% (22/99), 10.92% (25/229) vs. 4.44% (12/270), χ 2 = 27.37, 7.56, P < 0.05]. Positive CVB-IgG antibody results were detected in 7 counties. The overall positive rate of the population in the affected areas was higher than that of the control population [(4.09% (17/416) vs. 0.74% (2/270), χ 2 = 6.81, P = 0.009]. The positive rates of CVB-IgM and CVB-IgG antibodies in the population of the affected areas in Dayao County [22.22% (22/99), 9.80% (5/51)] were both higher than those of the control population ( P < 0.05). Among the five affected villages in Dayao County, the positive rates of CVB-IgM and CVB-IgG antibodies in the population of Aji Ju Village were the highest [25.49% (13/51), 3/13]. Conclusions:The positive rates of both CVB-IgM and CVB-IgG antibodies in the population of the areas affected by sudden death in Yunnan were higher than those of the control population, indicating that CVB infection occurred during the sudden death events in the above-mentioned affected areas.

Objective:This study investigated the awareness and consumption of Trogia venenata among populations in regions affected by Yunnan unexplained sudden death (YUSD). The findings aim to support etiological research on YUSD and contribute to the formulation of preventive measures against Trogia venenata poisoning. Methods:This study was a case-control study. From 2018 to 2021, surveys were conducted in 90 villages across 25 counties within YUSD-affected areas in Yunnan Province. Households with YUSD cases were designated as case households, whereas households without YUSD cases served as controls, ande were selected through convenience sampling at a 3:1 ratio. An enhanced questionnaire was designed to collect information on the consumption of Trogia venenata, and symptoms following consumption. Frequency data were presented as percentages, and group comparisons were conducted using χ 2 tests or Fisher’s exact tests. Results:A total of 711 questionnaires were collected (response rate: 100%), comprising 175 case households and 536 control households. Trogia venenata was present in 80.82% of the villages surveyed. Among the 711 households, 15.89% reported consuming Trogia venenata, primarily through stir-frying (53.10%), followed by boiling (29.20%), boiling and stir-frying (15.93%), and steaming (1.77%). Most households (94.69%) consumed fresh fruiting bodies, with 69.02% consuming them fewer than three times annually. The consumption rates were higher among the case households than among the control households. Of the 113 households with a history of Trogia venenata consumption, 35.40% reported symptoms such as nausea, vomiting, and limb soreness. The proportions of affected families in each group were compared according to their source, cooking method, fruiting body status and consumption frequency. The proportion of affected families with high consumption frequency (≥3 times/year) was higher than that with low consumption frequency (<3 times/year). Among 421 YUSD cases, 63 cases (14.96%) had a history of Trogia venenata consumption before death, with 43 cases showing symptoms within the longest known latency period (14 d) for poisoning by this mushroom. Conclusions:Trogia venenata is prevalent in 80.82% of YUSD-affected regions, with 16.67% of the population reporting its consumption, predominantly as fresh fruiting bodies prepared by stir-frying or boiling. Confirmed Trogia venenata consumption was identified in 14.96% of YUSD cases, suggesting that mushroom poisoning is a significant risk factor for YUSD. Ongoing health education and interventions are critical for mitigating the risk of Trogia venenata poisoning.

Objective:To analyze the infection status of Coxsackievirus B group (CVB) in regions affected by sudden unexplained death in Yunnan (referred to as sudden death in Yunnan), and to provide a scientific basis for formulating effective prevention and control strategies.Methods:A cross-sectional survey method was employed. The population from 16 counties (cities, districts, referred to as counties) affected by sudden death in Yunnan Province from 2002 to 2022 and the population from one non-affected county in 2021 and 2022 (control population) were classified into cases of sudden death in Yunnan (7 cases), co-occurring cases (29 cases), high-risk population (1 303 cases), and control population (270 cases). Blood samples were collected from these populations. By using enzyme-linked immunosorbent assay (ELISA), CVB immunoglobulin M (CVB-IgM) antibodies in the acute-phase serum samples of the population in the affected areas were detected, and CVB immunoglobulin G (CVB-IgG) antibodies in the convalescent-phase serum samples were detected. Both types of detections were carried out on the control population, and the test results were analyzed.Results:A total of 1 609 serum samples were tested, including 1 339 samples from the population in the affected areas (923 acute-phase samples and 416 convalescent-phase samples) and 270 samples from the control population. Among the 16 affected counties, positive CVB-IgM antibody results were detected in 9 counties. The overall positive rate of the population in the affected areas was higher than that of the control population [7.80% (72/923) vs. 4.44% (12/270), χ 2 = 40.78, P < 0.001]. The positive rates of the high-risk population in Dayao County and Lufeng City were both higher than that of the control population [(22.22% (22/99), 10.92% (25/229) vs. 4.44% (12/270), χ 2 = 27.37, 7.56, P < 0.05]. Positive CVB-IgG antibody results were detected in 7 counties. The overall positive rate of the population in the affected areas was higher than that of the control population [(4.09% (17/416) vs. 0.74% (2/270), χ 2 = 6.81, P = 0.009]. The positive rates of CVB-IgM and CVB-IgG antibodies in the population of the affected areas in Dayao County [22.22% (22/99), 9.80% (5/51)] were both higher than those of the control population ( P < 0.05). Among the five affected villages in Dayao County, the positive rates of CVB-IgM and CVB-IgG antibodies in the population of Aji Ju Village were the highest [25.49% (13/51), 3/13]. Conclusions:The positive rates of both CVB-IgM and CVB-IgG antibodies in the population of the areas affected by sudden death in Yunnan were higher than those of the control population, indicating that CVB infection occurred during the sudden death events in the above-mentioned affected areas.

Objective:To investigate the effect of stress-induced protein Sestrin2 (SESN2) on necroptosis of mouse dendritic cell (DC) induced by lipopolysaccharide (LPS) combined with zVAD, a panaspartate-specific cysteine protease (caspase) inhibitor.Methods:The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured. The cells were stimulated with LPS for 0 hour, 6 hours, 12 hours, and 24 hours, and grouped according to the stimulation time points. Western blotting was performed to determine the protein expression of SESN2 in each group. Overexpression empty lentivirus (NC), SESN2 gene overexpression RNA sequence lentivirus (SESN2 LV-RNA), small interfering empty lentivirus (NS), and SESN2 gene small interfering RNA sequence lentivirus (SESN2 siRNA) were transfected into DC2.4 cells. After 72 hours of transfection, cell fluorescence expression was observed under the inverted fluorescence microscope. Cells in each transfection group were stimulated with LPS for 24 hours. The blank control groups were set up and cultured with phosphate buffered saline (PBS) for 24 hours. Western blotting was performed to measure SESN2 protein expression. In the same groups as above, cells were stimulated with LPS+zVAD for 24 hours. The blank control groups were set up and cultured with PBS for 24 hours. Western blotting was used to determine the expression of mixed lineage kinase domain-like protein (MLKL) and phosphorylated-MLKL (p-MLKL). The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy. The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining.Results:Compared to the LPS 0 hour group, the expression of SESN2 in the LPS 24 hours group showed a significant increase. Therefore, 24 hours was chosen as the subsequent stimulation time point. After successful lentivirus transduction and 24 hours of cultivation, the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group. The MLKL phosphorylation in the SESN2 LV-RNA+LPS+zVAD group was significantly lower than that in the NC+LPS+zVAD group. The MLKL phosphorylation levels in both the NS+LPS+zVAD group and the NC+LPS+zVAD group were obviously higher than those in the NS+PBS group and the NC+PBS group, respectively. Laser scanning confocal microscopy showed that the trends in quantity and fluorescence intensity of p-MLKL protein expressions were consistent with the above results. The results from flow cytometry analysis and Hoechst staining showed that the rates of cell necrotic apoptosis in SESN2 siRNA+LPS+zVAD group were significantly higher than those in NS+LPS+zVAD group [flow cytometry analysis: (30.800±1.153)% vs. (20.800±1.114)%, Hoechst staining: (75.267±0.451)% vs. (46.267±3.371)%, both P < 0.05], indicating that knocking down SESN2 further exacerbated the occurrence of necroptosis. The necrotic apoptosis rates in SESN2 LV-RNA+LPS+zVAD group were significantly lower than those in NC+LPS+zVAD group [flow cytometry analysis: (7.160±0.669)% vs. (19.240±2.322)%, Hoechst staining: (32.433±3.113)% vs. (48.567±4.128)%, both P < 0.05], indicating that overexpressing SESN2 reversed such response and markedly reduced the proportion of necroptotic cells compared to the corresponding empty vector group. Conclusion:SESN2 exhibits an inhibitory effect on necroptosis of DC in sepsis. Targeted SESN2 expression may regulate the process of DC-mediated immune response in sepsis.

Objective:To investigate the associations of onset age, diabetes duration, and glycated hemoglobin (HbA1c) levels with ischemic stroke risk in type 2 diabetes patients.Methods:The participants were from Comprehensive Research on the Prevention and Control of the Diabetes in Jiangsu Province. The study used data from baseline survey from December 2013 to January 2014 and follow-up until December 31, 2021. After excluding the participants who had been diagnosed with stroke at baseline survey and those with incomplete information on onset age, diabetes duration, and HbA1c level, a total of 17 576 type 2 diabetes patients were included. Cox proportional hazard model was used to calculate the hazard ratio ( HR) and 95% CI of onset age, diabetes duration, and HbA1c level for ischemic stroke. Results:During the median follow-up time of 8.02 years, 2 622 ischemic stroke cases were registered. Multivariate Cox proportional risk regression model showed that a 5-year increase in type 2 diabetes onset age was significantly associated with a 5% decreased risk for ischemic stroke ( HR=0.95, 95% CI: 0.92-0.99). A 5-year increase in diabetes duration was associated with a 5% increased risk for ischemic stroke ( HR=1.05, 95% CI: 1.02-1.10). Higher HbA1c (per 1 standard deviation increase: HR=1.17, 95% CI: 1.13-1.21) was associated with an increased risk for ischemic stroke. Conclusion:The earlier onset age of diabetes, longer diabetes duration, and high levels of HbA1c are associated with an increased risk for ischemic stroke in type 2 diabetes patients.

Objective:To investigate the effects of stimulator of interferon gene (STING) on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 (ACSL4) in mouse dendritic cells (DCs) under sepsis, providing a basis for improving the dysregulation of immune response in sepsis caused by factors such as wound infection.Methods:This study was an experimental research. The mouse DC line DC2.4 in the logarithmic growth phase (with passages 3-10) were divided into lipopolysaccharide (LPS) stimulation 0 h (unstimulated) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 18 h group, and LPS stimulation 24 h group according to the random number table (the same grouping method below), which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of phosphorylated STING (p-STING), STING, and ACSL4 in cells were determined by Western blotting. DC2.4 successfully transfected with lentivirus containing STING gene small interfering RNA (hereinafter referred to as siSTING) were divided into siSTING+phosphate buffer solution (PBS) group and siSTING+LPS group. DC2.4 successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. After being stimulated with PBS or LPS and cultured for 24 hours, the protein expressions of p-STING, STING, and ACSL4 in cells were determined as above. Cell lipid peroxidation degrees were observed using the lipid peroxidation assay kit, and cell apoptosis rates were detected using flow cytometry. The sample numbers in the above cell experiments were all 3. Eighty male C57BL/6J mice aged 6 to 8 weeks were divided into sham surgery+normal saline (NS) group, cecal ligation and puncture (CLP)+NS group, sham surgery+C-176 group, and CLP+C-176 group, with 20 mice in each group. Mice in the two C-176 groups were intraperitoneally injected with C-176, while mice in the two NS groups were intraperitoneally injected with an equivalent volume of NS. One hour later, sham surgery was performed on the mice in the two sham surgery groups, and CLP surgery was performed on the mice in the two CLP groups to establish a sepsis model. At 24 h post-surgery, 10 mice from each group were sacrificed to extract spleen DCs, and protein expression, lipid peroxidation, and apoptosis rates were detected as above ( n=3). Hematoxylin-eosin staining was performed to observe pathological damage in the heart, liver, lung, and kidney tissue. The remaining 10 mice in each group were observed for survival within 7 days after surgery. Results:The protein expressions of p-STING, STING, and ACSL4, as well as the p-STING/STING ratio in DC2.4 in LPS stimulation 24 h group were significantly higher than those in LPS stimulation 0 h group ( P<0.05). After 24 h of culture, the protein expressions of p-STING, STING, and ACSL4 in DC2.4 in siSTING+LPS group and empty vector+PBS group were significantly lower than those in empty vector+LPS group ( P<0.05); the lipid peroxidation degrees of DC2.4 in siSTING+LPS group and empty vector+PBS group were weaker than those in empty vector+LPS group. The apoptosis rates of DC2.4 in empty vector+PBS group, empty vector+LPS group, siSTING+PBS group, and siSTING+LPS group were (15.7±3.0)%, (37.8±2.9)%, (13.1±2.1)%, and (20.6±1.8)%, respectively. The apoptosis rates of DC2.4 in empty vector+PBS group and siSTING+LPS group were significantly lower than that in empty vector+LPS group ( P<0.05). At 24 h post-surgery, the protein expressions of p-STING and ACSL4, and the p-STING/STING ratio in spleen DCs of mice in CLP+NS group were significantly higher than those in sham surgery+NS group and CLP+C-176 group ( P<0.05); the protein expression of STING in spleen DCs of mice in CLP+NS group was significantly higher than that in sham surgery+NS group ( P<0.05); the lipid peroxidation degrees of spleen DCs of mice in CLP+C-176 group and sham surgery+NS group were weaker than that in CLP+NS group. The apoptosis rates of spleen DCs of mice in sham surgery+NS group and CLP+C-176 group were significantly lower than that in CLP+NS group ( P<0.05), and the apoptosis rate of spleen DCs of mice in CLP+C-176 group was significantly higher than that in sham surgery+C-176 group ( P<0.05). Pathological tissue damage in the heart, liver, lung, and kidney of mice in CLP+NS group was significantly worse than that in sham surgery+NS group, while such damage in the above organs of mice in CLP+C-176 group was significantly alleviated compared with that in CLP+NS group. The survival ratio of mice in CLP+NS group within 7 days after surgery was significantly lower than that in sham surgery+NS group ( χ2=8.30, P<0.05). Conclusions:Under sepsis, STING activation in mouse DCs is significant, which enhances ACSL4-mediated ferroptosis. Inhibiting STING activation can significantly reduce ACSL4-mediated ferroptosis level in mouse DCs under sepsis, thereby improving the survival rate of septic mice.

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
Humans ; Apoptosis Inducing Factor/metabolism* ; NAD/metabolism* ; Dimerization ; Apoptosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with unclear etiology and limited treatment options. The median survival time for IPF patients is approximately 2-3 years and there is no effective intervention to treat IPF other than lung transplantation. As important components of lung tissue, endothelial cells (ECs) are associated with pulmonary diseases. However, the role of endothelial dysfunction in pulmonary fibrosis (PF) is incompletely understood. Sphingosine-1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor highly expressed in lung ECs. Its expression is markedly reduced in patients with IPF. Herein, we generated an endothelial-conditional S1pr1 knockout mouse model which exhibited inflammation and fibrosis with or without bleomycin (BLM) challenge. Selective activation of S1PR1 with an S1PR1 agonist, IMMH002, exerted a potent therapeutic effect in mice with bleomycin-induced fibrosis by protecting the integrity of the endothelial barrier. These results suggest that S1PR1 might be a promising drug target for IPF therapy.

ObjectiveTo investigate the mechanism of Xiaonang Tiaojing decoction（XNTJD）in improving polycystic ovary syndrome with insulin resistance（PCOS-IR）model rats based on anti-Müllerian hormone（AMH）/AMH type Ⅱ receptor（AMHRⅡ）signaling pathway. MethodForty-eight adult female SD rats were randomly divided into the blank group， model group， XNTJD group（11.4 g·kg^-1·d^-1） and Diane-35 group（0.21 g·kg^-1·d^-1）， PCOS-IR model was established by high-fat and high-sugar diet combined with letrozole in rats of all groups except the blank group， rats in the administration groups were given the corresponding dose of drugs by gavage for 15 days with an interval of 1 d every 4 d， normal saline of the same volume was given to the blank group and the model group. Estrous cycle was recorded daily during treatment. At the end of treatment， body weight and Lee's index were recorded， AMH， luteinizing hormone（LH）， LH/follicle stimulating hormone（FSH）， testosterone（T）and estradiol（E₂）levels were measured by enzyme-linked immunosorbent assay（ELISA）， fasting plasma glucose（FPG）was measured by glucometer， fasting insulin（FINS） level was measured by radioimmunoassay（RIA）， and the insulin resistance index（HOMA-IR） and insulin sensitivity index（QUICKI）were calculated， triglyceride（TG）and total cholesterol（TC）levels were measured by automatic biochemical analyzer， hematoxylin-eosin（HE）staining was used to observe the morphological changes of the ovary， the levels of AMHRⅡ， bone morphogenetic protein-15（BMP-15）and Smad5 in ovarian tissues were detected by immunohistochemistry（IHC），Western blot was used to analyze the protein expression levels of AMHRⅡ， BMP-15 and Smad5. ResultCompared with the blank group， a large number of leukocytes were observed in the vaginal exfoliated cells of rats in the model group， mainly in diestrus， the levels of body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）were significantly increased（P<0.01）， and QUICKI and E₂ levels were significantly decreased（P<0.01）， there were more cystic bulges on the ovarian surface， more wet weight， more atretic and cystic dilated follicles in the ovarian tissues， and the thickness of granulosa cell layer was reduced without oocytes， the expression level of AMHRⅡ protein in ovarian tissues was significantly increased（P<0.01）， and the expression levels of BMP-15 and Smad5 proteins were significantly decreased（P<0.01）. Compared with the model group， the exfoliated cells in the vagina of rats treated with XNTJD group showed keratinocytes from the 5^th to 6^th day of treatment， and a stable estrous cycle gradually appeared， body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）levels were significantly decreased（P<0.05， P<0.01）， QUICKI and E₂ levels were significantly increased（P<0.01）， ovarian surface was smoother and lighter in wet weight， oocytes and mature follicles were observed in ovarian tissues， the thickness of granulosa cell layer increased and the morphology was intact， the expression levels of BMP-15 and Smad5 proteins were significantly increased（P<0.01）and the expression level of AMHRⅡ protein was significantly decreased（P<0.01）in ovarian tissues. ConclusionXNTJD may mediate the up-regulation of BMP-15 and Smad5 in ovarian tissues of PCOS-IR rats by down-regulating AMH/AMHRⅡ， thereby improving ovarian function， sex hormones and glucose-lipid metabolism levels in PCOS-IR rats.

Objective:To evaluate the effect of comprehensive intervention measures on Yunnan unexplained sudden death (YUSD) in Dali Prefecture, Yunnan Province, and to provide scientific basis for improving the prevention and control measures.Methods:Since 2010, Yunnan Province had implemented comprehensive intervention measures in ward areas according to the etiological pattern of YUSD. In July 2019, 47 families with YUSD were selected as case families and 23 families without YUSD were selected as control families in 31 natural villages of Heqing, Xiangyun, Yunlong, Eryuan, Jianchuan, Binchuan and Nanjian counties of Dali Prefecture. A unified questionnaire was used to investigate the basic information, economic status, dietary structure, and health literacy of the families during the two periods of "the first sudden death case" and "the present".Results:The annual household income of the case families at present (median, 20 492.6 yuan) was significantly higher than that of the first sudden death case (3 883.4 yuan), and the difference was statistically significant ( Z = - 5.27, P < 0.001). At present, rice (76.6%, 36/47) was the main diet of the case families; at the time of the first sudden death case, 23.4% (11/47) of the case families could not eat enough, and there was no such situation in the case families at present. Compared with the time of the first sudden death case, the dietary habits of the case families at present were as follows: the proportion of eating Trogia venenata decreased from 19.0% (39/205) to 0 (0/190), the proportion of eating wild fruit decreased from 17.1% (35/205) to 9.5% (18/190), and the proportion of drinking raw water decreased from 55.1% (113/205) to 42.1% (80/190), and the differences were statistically significant (χ 2 = 22.37, 4.90, 6.86, P < 0.05). Lifestyle and health awareness: the proportion of those who washed their hands before meals and after using the toilet increased from 9.8% (20/205) to 41.6% (79/190), those who did not overwork increased from 16.6% (34/205) to 34.2% (65/190), and those who took good protection when spraying pesticides increased from 7.3% (15/205) to 21.6% (41/190), and the differences were statistically significant (χ 2 = 53.17, 33.94, 16.48, P < 0.001). Toilet habits: the proportion of using outdoor toilet decreased from 75.6% (155/205) to 9.5% (18/190), the difference was statistically significant (χ 2 = 175.21, P < 0.001). When the first sudden death case occurred, the proportions of eating Trogia venenata and using outdoor toilet in the case families were higher than those in the control families (χ 2 = 22.37, 23.70, P < 0.001), the proportions of those who washed their hands before meals and after using the toilet and those who did not overwork in the case families were lower than those in the control families (χ 2 = 7.38, 4.93, P < 0.05). Conclusions:The economic conditions, production and living conditions of YUSD areas in Dali Prefecture have been significantly improved, and the health literacy and health prevention awareness of the population have been greatly improved. Economic conditions and living standard, dietary structure and health literacy may be related factors of YUSD.