A novel analytical method was developed in this study by combining online solid phase extraction with ultra performance liquid chromatography-tandem mass spectrometry(Online SPE-UPLC-MS/MS)for simultaneous determination of 50 kinds of steroid hormones in surface water.Specifically,after high-speed centrifugation of 4 mL water samples,the supernatant was directly injected into an Oasis HLB online SPE column for enrichment and purification.Subsequently,the target compounds were transferred to the analytical column via valve switching for separation and analysis.The chromatographic separation was performed on a Thermo Acclaim RSLC C18 column(100 mm×2.1 mm,2.2 μm),using a mobile phase composed of 5 mmol/L ammonium fluoride aqueous solution and acetonitrile.Mass spectrometric detection was conducted in positive ion mode,utilizing multiple reaction monitoring(MRM)with quantification achieved by the internal standard method.The method validation demonstrated that the limits of detection(LOD)for the 50 kinds of steroid hormones ranged from 0.02 to 0.50 ng/L,while the limits of quantification(LOQ)were between 0.08 and 1.67 ng/L.The average recoveries in surface water samples at spiked concentrations of 5,20 and 200 ng/L were between 74.1％and 119％,with relative standard deviations(RSDs)of 0.2％to 9.9％.This method was applied to analyze 11 surface water samples collected from sites surrounding a pharmaceutical and chemical industrial park.A total of 44 kinds of steroid hormones were detected,with concentrations ranging from 0.11 to 88.6 ng/L,revealing the presence of hormone contamination in the environmental waters surrounding industrial areas.Compared with the traditional offline SPE methods,the proposed online SPE technique significantly reduced sample volume requirements and pretreatment time,while minimizing the loss of target compounds during the pretreatment process.Moreover,compared to reported online SPE techniques,this method achieved high-throughput analysis of multiple classes of steroid hormones,with lower detection limits and higher recoveries.Overall,this method provided rapid sample preparation,high sensitivity,and excellent stability,making it suitable for the direct analysis of trace steroid hormones in surface water.

Objective To study the pathogenic bacteria infection in hospitalized diabetic foot patients in the Third People's Hospital of Yunnan Province and its correlation with different Wagner grades,to understand the the characteristics of pathogenic bacteria and related risk factors in hospitalized diabetic foot patients in the Third People's Hospital of Yunnan Province,and to further provide theoretical guidance for anti-infection treatment of these patients.Methods A retrospective analysis was conducted on the demographic data,severity of foot ulcers,and related laboratory test results of 536 patients with diabetic foot who were detected to have bacterial infection in the Third People's Hospital of Yunnan Province from January 2019 to January 2023.Results Among the 536 diabetic foot patients,pathogenic bacteria were cultured from 268 cases(50.0%)of Gram-positive bacterial infections,214 cases(39.9%)of gram-negative bacterial infections,2 cases(0.4%)of fungal infections,and 52 cases(9.7%)of mixed bacterial infections.The main pathogens among gram-positive bacteria were Staphylococcus aureus,Staphylococcus epidermidis and Enterococcus faecalis.for Gram-negative bacteria,the main pathogens were Escherichia coli,Enterobacter cloacae and Klebsiella pneumoniae.There were 31 cases of multi-drug resistant bacteria,and the multi-drug resistance rate was(5.78%).Among Gram-positive bacteria,all multidrug-resistant strains were Staphylococcus aureus,while among Gram-negative bacteria,the multi-drug resistant strains included Acinetobacter baumannii(1 case),Klebsiella pneumoniae(2 cases),Proteus common(2 cases),Pseudomonas aeruginosa(5 cases),Proteus mirabilis(1 case)and Enterobacter cloacae(1 case).The 536 patients were divided into Wagner grade 1 and 2 groups(78 cases),Wagner grade 3 group(274 cases),and Wagner grade 4 and 5 groups(184 cases).There were 73 cases of single bacterial infections and 5 cases of mixed bacterial infections in Wagner grade 1 and 2 group,including 51 cases(65.4%)of gram-positive bacteria,21 cases(26.9%)of gram-negative bacteria and 1 case(1.3%)of fungi.There were 248 cases of single bacterial infections and 26 cases of mixed bacterial infections in Wagner3 group,with 144 cases(52.6%)of gram-positive bacteria,103 cases(37.6%)of gram-negative bacteria,and 1 case(0.4%)with fungi.In the Wagner grade 4 and 5 groups,there were 163 cases of single bacterial infections and 21 cases of mixed bacterial infection,with 73 strains(39.7%)of gram-positive bacteria,90 strains(48.9%)of gram-negative bacteria and 0 strain(0%)of fungi.The predominant infectious pathogens in Wagner grades 1,2 and 3 were gram-positive bacteria,while those in Wagner grades 4 and 5 patients were mainly gram-negative bacteria.There were statistically significant differences in white blood cell counts,neutrophil percentage,bacterial classification,length of hospital stay,erythrocyte sedimentation rate and albumin levels among diabetic foot patients with different Wagner grades(P<0.01).With the increase of Wagner grade,patients had higher white blood cell counts and hypersensitive C-reactive protein levels,longer hospital stays,and lower albumin levels;however,there were no statistically significant differences in age,sex,duration of diabetes,smoking history,alcohol consumption history and history of hypertension(P>0.05).Conclusion The bacterial infection situation in patients with diabetic foot ulcers is related to different Wagner grades.The higher the Wagner grades,the greater the likelihood of infection with gram-negative bacteria.Antibiotics can be reasonably selected according to the Wagner grades of patients upon admission,actively controlling infection,while also enhancing,shortening hospital stays,and reducing amputation rates,thereby improving the prognosis of diabetic foot patients.

Objective To analyze the bacterial distribution characteristics,drug resistance characteristics and related risk factors of multidrug-resistant organisms(MDRO)in patients with diabetic foot infection(DFI)in some areas of Yunnan Province to provide empirical reference for clinical treatment.Methods Clinical data of 300 DFI patients admitted to the Department of Endocrinology of the Third People's Hospital of Yunnan Province from January 2019 to December 2023 were collected.Based on the results of drug sensitivity tests and matching of basic data,patients were divided into the MDRO group(n=60)and the non-MDRO group(n=240).A retrospective analysis was conducted on the distribution of pathogenic bacteria,drug resistance characteristics of MDRO and risk factors for MDRO infection in DFI patients.Results In 60 patients with MDRO infections,62 strains of MDRO were cultured,with 58 strains from single MDRO infections and 4 strains from mixed MDRO infections.Of the 60 patients,2 were cultured for 2 types of MDRO.Among the strains,there were 45 gram-positive bacteria(72.58％)which were all Staphylococcus aureus,17 strains of gram-negative bacteria(27.42％)mainly including Pseudomonas aeruginosa,Enterobacter cloacae and Klebsiella pneumoniae.Among common MDRO,Staphylococcus aureus showed complete resistance to penicillin G and oxacillin(100％),with high resistance to erythromycin and clindamycin(＞80％),but no resistance to tigacycline vancomycin was observed.The resistance of Klebsiella pneumoniae and Enterobacter cloacae to cephalosporin antibiotics was obvious,and the resistance rate to imipenem and amikacin was low.Pseudomonas aeruginosa was 100％resistant to ticacillin/clavulanate potassium,imipenem,tigacycline and cotrimoxazole,but showed no resistance to cefepime,ciprofloxacin,gentamicin and amikacin.There were statistically significant differences between the two groups in regional distribution,duration of diabetic foot,lower extremity arterial disease,venous plasma glucose levels and glycosylated hemoglobin(P＜0.05).Binary Logistic regression analysis showed that region and duration of diabetic foot disease were independent risk factors for MDRO infection in DFI patients(P＜0.05).Conclusion In some areas of Yunnan Province,the distribution of MDRO in DFI patients is mainly gram-positive bacteria,with varying antibiotic sensitivities among different pathogens.Multiple factors lead to MDRO infections in DFI patients,which assists clinical practitioners in early identification of high-risk DFI patients with MDRO infections and provide empirical reference for clinical treatment.

Aim To study the neuroprotective effects of Herba siegesbeckiae extract on cerebral ischemia/ reperfusion rats and its mechanism. Methods Sixty SD rats were randomly divided into model group, low, middle and high dose groups of Herba siegesbeckiae, and Sham operation group, and the drug was given continuously for seven days. The degree of neurologic impairment was evaluated by mNSS, and the infarct volume was measured by MRI. The number of Nissl-posi- tive cells was detected by Nissl staining, and the apop- tosis was accessed by Tunel staining. Furthermore, the expression of Bax, Bcl-2 and NeuN was observed by Western blot, and the expression of NeuN was detected by immunofluorescence staining. The expression of IL- 1β, TNF-α and IL-6 mRNA was performed by RT- qPCR. Results The mNSS score and the volume of ischemic cerebral infarction in the model group were significantly increased, and Herba siegesbeckiae extract treatment significantly decreased the mNSS score and infarct volume (P<0.05, P<0.01). Herba siegesbeckiae extract could increase the number of Nissl-pos- itive cells and the expression of NeuN (P<0.01), and reduce the number of Tunel-positive cells (P<0.01). Western blot showed that Herba siegesbeckiae extract inhibited the expression of Bax, increased Bcl-2 and NeuN in ischemic brain tissue (P<0.01). RT-qPCR showed that Herba siegesbeckiae extract inhibited the expression of IL-1 β, TNF-α and IL-6 mRNA in the is-chemic brain tissue (P<0.01). Conclusions Herba siegesbeckiae extract can reduce the cerebral infarction volume, improve the neurological function damage, inhibit the apoptosis of nerve cells and the expression of inflammatory factors and promote the expression of NeuN, there by exerting protective effects on MCAO rats.

Aim To study the permeability of salidro-side(Sal)to the blood brain barrier(BBB)by high-performance liquid chromatography electrospray ioniza-tion tandem mass spectrometry(UPLC-ESI-MS-MS),and to explore the target and mechanism of Sal in the treatment of ischemic stroke(IS)by network pharma-cology,molecular docking technique and animal exper-iment.Methods UPLC-ESI-MS/MS was used to study the BBB penetration of Sal.Multiple databases were used to predict the target of Sal and the disease target of IS,GO and KEGG enrichment analysis were performed and verified by molecular docking technique and animal experiments.Results After Sal adminis-tration to normal rats and MCAO rats,Sal prototype and the metabolite tyrosol were detected in plasma and brain tissue of rats.A total of 191 targets were identi-fied by network pharmacology,the enrichment analysis of GO mainly involved in the biological processes of proteolysis and positive regulation of cell migration,and the analysis of KEGG pathway suggested that PI3K-Akt,MAPK,FOXO and other signaling path-ways played a key role in the treatment of IS by Sal The results of molecular docking showed that Sal had good binding ability with the core target of docking,and the results of animal experiments showed that Sal could significantly improve the neurologic impairment of MCAO rats,the number of Nissl-positive cells in is-chemic side significantly increased,and the expression of VEGF,EGFR and IGF1 increased,while the ex-pression of IL-6 and MMP9 was inhibited.Conclu-sions Sal is able to penetrate the BBB and enter the central nervous system for its pharmacological effects.Network pharmacology predicts the core targets of Sal in the treatment of IS,including VEGFA,EGFR,IL-6,MMP9,IGF1,CASP3,ALB,SRC.The effects of Sal on some core targets can be verified by animal ex-periments,to provide a reference for further study of the mechanism of Sal in the treatment of IS.

The function of the central nervous system was significantly altered under high-altitude hypoxia, and these changes lead to central nervous system disease and affected the metabolism of drugs in vivo. The blood-brain barrier is essential for maintaining central nervous system stability and plays a key role in the regulation of drug metabolism, and barrier structure and dysfunction affect drug transport to the brain. Changes in the structure and function of the blood-brain barrier and the transport of drugs across the blood-brain barrier under high-altitude hypoxia are regulated by changes in brain microvascular endothelial cells, astrocytes and pericytes, and are regulated by drug metabolism factors such as drug transporters and drug metabolizing enzymes. This article reviews the effects of high-altitude hypoxia on the structure and function of the blood-brain barrier and the effects of changes in the blood-brain barrier on drug metabolism. We investigate the regulatory effects and underlying mechanisms of the blood-brain barrier and related pathways such as transcription factors, inflammatory factors and nuclear receptors on drug transport under high-altitude hypoxia.

Aim To study the effect of salidroside (SAL) on cerebral vascular endothelial cells of rats with ischemic brain injury and its mechanism of action. Methods Twenty-four healthy adult SD male rats were prepared by bolt plugging method to prepare MCAO models,and randomly divided into sham surgery group ( Sham ) , model group ( MCAO ) , and SAL administration group (MCAO + SAL) ,and the concentration of SAL was 50 mg • kg ~ , with a continuous administration for six days. Western blot was used to detect the protein expression of ICAM-1, VCAM-1 , E-se-lectin,and P-selectin in injured brain tissue of rats. In vitro cell experiments using HUVECs were subjected to different concentrations of salidroside (0. 1,1,10 jjunol • L ) and LPS (100 ^g • L ) intervened for 24 hours,and CCK-8 was employed to detect the effects of SAL and LPS on the survival of HUVECs. In vitro an-giogenesis experiments, LPS group ( 100 (jLg • L~ ) and SAL administration group ( LPS + Sal) intervened in HUVECs for 24 hours,and the concentrations of SAL administration were 0. 1,1, and 10 jjunol • L , then the effects of LPS and SAL on their angiogenesis were observed. The protein expressions of ICAM-1, VCAM-1 ,E-selectin,and P-selectin in HUVECs were detected by Western blot. Results SAL could reduce the expression of ICAM-1, VCAM-1, E-selectin, and P-selectin in ischemic brain tissue of MCAO rats. In vitro experimental studies found that salidroside had no effect on the survival of HUVECs. LPS inhibited the angiogenesis of HUVECs, and after the action of SAL, SAL (1,10 jjimol • L ) reversed the effect of LPS and promoted its angiogenesis. Compared with the control group,the expressions of ICAM-1, VCAM-1, E-selectin and P-selectin of HUVECs after LPS stimulation increased, while the expressions of ICAM-1, VCAM-1 , E-selectin and P-selectin were significantly reduced after the addition of SAL, which promoted the angiogenesis ability of HUVECs. Conclusions SAL can improve the ability of cell regeneration in rats with ischemic brain injury and promote the ability of blood vessel formation.

Aim To develop a ultra-high performance liquid chromatography electrospray-ionization tandem mass spectrometry ( UPLC-MS/MS ) method for the simultaneous determination of salidroside derivative pOBz in rat plasma and brain tissue, and to study the pharmacokinetic profile and penetration of the blood-brain barrier in rats after a single dose intravenous administration of pOBz. Methods SD rats were administered pOBz at a dose of 50 mg • kg

Aim To investigate the neuroprotective effect of prophylactic administration of salidroside (Sal) on MCAO rats. Methods A total of 52 SD adult male rats were randomly divided into sham operation group (Sham), model group (MCAO) and salidroside pre-administration group (MCAO + Sal). The dose of Sal was 50 mg·kg