ObjectiveTo explore the possible mechanisms by which ligustilide （LIG） exerts neuroprotective effects on ischemic stroke （IS） by inhibiting the release of neutrophil extracellular traps （NETs）， promoting blood-brain barrier repair， and alleviating post-ischemic neuroinflammation， thereby providing a new direction for IS treatment. MethodsA middle cerebral artery occlusion （MCAO） model was established in rats. The rats were divided into the sham operation （Sham） group， model （Model） group， low- and high-dose LIG groups （20， 40 mg·kg^-1）， and the NET inhibitor CI-amidine group （CI-amidine， 10 mg·kg^-1）. Drug treatments were administered for 3 days. Neurological injury after ischemia was evaluated by 2，3，5-triphenyltetrazolium chloride （TTC） staining， neurological deficit scoring， and brain index measurement. Flow cytometry and Western blot were used to analyze changes in neutrophil expression. Immunofluorescence was used to observe the fluorescence intensity of the NET marker citrullinated histone H3 （H3Cit）. Western blot was performed to detect the expression of blood-brain barrier tight junction-related proteins and inflammatory factors， including interleukin-18 （IL-18） and interleukin-1β （IL-1β）. ResultsCompared with the Sham group， the Model group exhibited significant brain tissue injury （P<0.05）， significantly increased neutrophil numbers and NET expression （P<0.05）， significantly impaired blood-brain barrier permeability （P<0.05）， and significantly increased expression of inflammatory factors （P<0.05）. Compared with the Model group， both low- and high-dose LIG significantly alleviated brain tissue injury in rats （P<0.01）， inhibited neutrophil numbers and NET expression （P<0.01）， reduced blood-brain barrier damage （P<0.01）， and suppressed the expression of inflammatory factors IL-18 and IL-1β （P<0.01）， thereby ultimately exerting a neuroprotective effect. ConclusionThe neuroprotective effect of LIG in rats with cerebral ischemia-reperfusion injury may be related to inhibition of neutrophils and the NETs induced by them.

ObjectiveTo explore the possible mechanisms by which ligustilide （LIG） exerts neuroprotective effects on ischemic stroke （IS） by inhibiting the release of neutrophil extracellular traps （NETs）， promoting blood-brain barrier repair， and alleviating post-ischemic neuroinflammation， thereby providing a new direction for IS treatment. MethodsA middle cerebral artery occlusion （MCAO） model was established in rats. The rats were divided into the sham operation （Sham） group， model （Model） group， low- and high-dose LIG groups （20， 40 mg·kg^-1）， and the NET inhibitor CI-amidine group （CI-amidine， 10 mg·kg^-1）. Drug treatments were administered for 3 days. Neurological injury after ischemia was evaluated by 2，3，5-triphenyltetrazolium chloride （TTC） staining， neurological deficit scoring， and brain index measurement. Flow cytometry and Western blot were used to analyze changes in neutrophil expression. Immunofluorescence was used to observe the fluorescence intensity of the NET marker citrullinated histone H3 （H3Cit）. Western blot was performed to detect the expression of blood-brain barrier tight junction-related proteins and inflammatory factors， including interleukin-18 （IL-18） and interleukin-1β （IL-1β）. ResultsCompared with the Sham group， the Model group exhibited significant brain tissue injury （P<0.05）， significantly increased neutrophil numbers and NET expression （P<0.05）， significantly impaired blood-brain barrier permeability （P<0.05）， and significantly increased expression of inflammatory factors （P<0.05）. Compared with the Model group， both low- and high-dose LIG significantly alleviated brain tissue injury in rats （P<0.01）， inhibited neutrophil numbers and NET expression （P<0.01）， reduced blood-brain barrier damage （P<0.01）， and suppressed the expression of inflammatory factors IL-18 and IL-1β （P<0.01）， thereby ultimately exerting a neuroprotective effect. ConclusionThe neuroprotective effect of LIG in rats with cerebral ischemia-reperfusion injury may be related to inhibition of neutrophils and the NETs induced by them.

OBJECTIVE: To explore the effect mechanism of electroacupuncture (EA) for ameliorating diabetic peripheral neuropathy (DPN) based on the analysis of gut microbiota and metabolomics. METHODS: Thirty SPF-grade male SD rats were randomly divided into a normal group, a model group, and an EA group, with 10 rats in each one. Except in the normal group, the intraperitoneally injection with streptozotocin was used to induce diabetes mellitus model in the rest groups. In the EA group, acupuncture was delivered at bilateral "Zusanli" (ST36), "Sanyinjiao" (SP6), "Pishu" (BL20) and "Shenshu" (BL23), and electric stimulation was attached to "Zusanli" (ST36)-"Sanyinjiao" (SP6) and "Pishu" (BL20)-"Shenshu" (BL23), on the same side, with continuous wave and a frequency of 2 Hz, for 10 min in each intervention. The intervention measure of each group was delivered once every 2 days, 3 times a week, for 8 consecutive weeks. Body weight, random blood glucose (RBG), thermal withdrawal latency (TWL), and mechanical withdrawal threshold (MWT) before intervention, and in 4 and 8 weeks of intervention, separately, as well as sensory nerve conduction velocity (SCV) and motor nerve conduction velocity (MCV) of the sciatic nerve after intervention were measured. Metagenomic sequencing (MS) was used to analyze gut microbiota and screen for differential species. Liquid chromatography-mass spectrometry (LC-MS) was employed to detect the differential metabolites in plasma, and the metabolic pathway enrichment analysis was performed on the differential metabolites. Spearman correlation analysis was adopted to assess the relationship between gut microbiota and metabolomics. RESULTS: After 4 and 8 weeks of intervention, when compared with the model group, the EA group showed the increase in body weight, TWL, MWT (P<0.01), and the decrease in RBG (P<0.01). Compared with the normal group, SCV and MCV, as well as Chao1 index were dropped in the model group (P<0.01), and those were elevated in the EA group when compared with those in the model group (P<0.01). The dominant bacterial phyla of each group were Firmicutes (F) and Bacteroidota (B), the ratio of them (F/B) in the model group was lower than that of the normal group (P<0.05), and F/B in the EA group was higher when compared with that in the model group (P<0.05). In comparison with the normal group, the relative abundance increased in Prevotella, Segatella, Prevotella-hominis and Segatella-copri (P<0.05); and it decreased in Ligilactobacillus, Eubacterium, Pseudoflavonifractor, Ligilactobacillus-murinus (P<0.05) in the model group. Compared with the model group, the relevant abundance of the above mentioned gut bacteria was all ameliorated in the EA group (P<0.05, P<0.01). Among the three groups, 120 differential metabolites were identified and enriched in 28 key metabolic pathways, such as glycerophospholipid and linoleic acid, of which, glycerophospholipid was the most significantly affected pathway in EA intervention. Spearman correlation analysis showed that 6 phosphatidylcholine metabolites were significantly positively correlated with Pseudoflavonifractor and were negatively with Prevotella, Segatella, Prevotella-hominis, Segatella-copri; 5 phosphatidylethanolamine metabolites were significantly negatively correlated with Pseudoflavonifractor and positively correlated with Prevotella, Segatella, Prevotella-hominis, Segatella-copri. CONCLUSION EA may regulate metabolic pathways such as glycerophospholipid, modulate specific gut microbiota such as Pseudoflavonifractor, Prevotella, and Segatella, and the co-expressed differential metabolites like phosphatidylcholine and phosphatidylethanolamine, thereby reducing blood glucose and protecting nerve function, so as to relieve the symptoms of DPN of rats.
Animals ; Electroacupuncture ; Male ; Gastrointestinal Microbiome ; Diabetic Neuropathies/microbiology* ; Rats, Sprague-Dawley ; Rats ; Metabolomics ; Humans ; Acupuncture Points

OBJECTIVES: To explore the molecular mechanism by which lncRNA SNHG15 regulates proliferation, invasion and migration of lung adenocarcinoma cells. METHODS: The lncRNA microarray chip dataset GSE196584 and LncBase were used to predict the lncRNAs that interact with miR-30b-3p, and their association with patient prognosis were investigated using online databases, after which lncRNA nucleolar RNA host gene 15 (SNHG15) was selected for further analysis. The subcellular localization of lncRNA SNHG15 and its expression levels in normal human lung epithelial cells and lung adenocarcinoma cell lines were detected using fluorescence in situ hybridization and qRT-PCR. In cultured A549 cells, the changes in cell proliferation, migration, and invasion following transfection with a SNHG15 knockdown plasmid (sh-SNHG15), a miR-30b-3p inhibitor, or their co-transfection were assessed with EdU, wound healing, and Transwell assays. Bioinformatics analyses were used to predict the regulatory relationship between lncRNA SNHG15 and COX6B1, and the results were verified using Western blotting and rescue experiments in A549 cells transfected with sh-SNHG15, a COX6B1-overexpressing plasmid, or both. RESULTS: LncRNA SNHG15 was shown to target miR-30b-3p, and the former was highly expressed in lung adenocarcinoma, and associated with a poor patient prognosis. LncRNA SNHG15 was localized in the cytoplasm and expressed at higher levels in A549 and NCI-H1299 cells than in BEAS-2B cells. In A549 cells, lncRNA SNHG15 knockdown significantly inhibited cell migration, invasion and proliferation, and these changes were reversed by miR-30b-3p inhibitor. A regulatory relationship was found between lncRNA SNHG15 and COX6B1, and their expression levels were positively correlated (r=0.128, P=0.003). MiR-30b-3p knockdown obviously decreased COX6B1 expression in A549 cells, and COX6B1 overexpression rescued the cells from the inhibitory effects of lncRNA-SNHG15 knockdown. CONCLUSIONS LncRNA SNHG15 may compete with COX6B1 to bind miR-30b-3p through a ceRNA mechanism to affect proliferation, migration, and invasion of lung adenocarcinoma cells.
Humans ; MicroRNAs/metabolism* ; RNA, Long Noncoding/genetics* ; Cell Proliferation ; Cell Movement ; Lung Neoplasms/genetics* ; Adenocarcinoma of Lung ; Neoplasm Invasiveness ; A549 Cells ; Adenocarcinoma/genetics* ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor

OBJECTIVES: To elucidate the anti-aging effect of β-sitosterol (BS), an important component in the fruits of Alpinia oxyphylla Miq., in C. elegans and its regulatory effect on ETS-5 gene to modulate ferroptosis. METHODS: C. elegans treated with 10 µg/mL BS were monitored for survival time and changes in body length, motility, and reproductive function. The effect of ETS-5 gene knockdown on survival time of C. elegans was observed, and the changes in fat accumulation and lipid redox homeostasis in the transfected C. elegans were assessed using Oil Red O staining and by detecting MDA levels and the GSH/GSSG ratio. The mRNA expression levels of ferroptosis-related genes (FTN-1, GPX-1 and AAT-9) were detected using qPCR. The effects of BS treatment and ETS-5 knockdown on AAT-9 enzyme activity in C. elegans were examined. The effect of BS on nuclear localization of FEV (the human homolog of ETS-5) was validated in cultured human umbilical venous endothelial cells (HUVECs). RESULTS: Both BS treatment and ETS-5 knockdown significantly prolonged the lifespan, promoted lipid accumulation and reduced lipid peroxidation in C. elegans. ETS-5 knockdown resulted in upregulated expressions of the ferroptosis repressors GPX-1, AAT-9 and FTN-1 and increased the GSH/GSSG ratio in C. elegans. CONCLUSIONS BS inhibits ferroptosis in C. elegans by suppressing the expression of ETS-5 transcription factor and hence the activity of AAT-9 enzyme, a key gene for ferroptosis, which in turn prolongs the lifespan of C. elegans.
Animals ; Caenorhabditis elegans/physiology* ; Ferroptosis/drug effects* ; Alpinia/chemistry* ; Sitosterols/pharmacology* ; Longevity/drug effects* ; Fruit/chemistry* ; Humans

ObjectiveTo explore the quantity-quality transfer process of medicinal materials， decoction pieces and standard decoction of Haliotidis Concha（Haliotis discus hannai） by analyzing the physical phase and compositional changes， so as to provide references for the effective control of its quality. MethodsA total of 20 batches of Haliotidis Concha（H. discus hannai） from different habitats were collected and prepared into corresponding calcined products and standard decoction， and the content of CaCO₃ of the three samples were determined and the extract yield and transfer rate of CaCO₃ were calculated. The changes in elemental composition and their relative contents were investigated by X-ray fluorescence spectrometry（XRF）， X-ray diffraction（XRD） was used to study the changes in the phase compositions of the three samples and to establish their respective XRD specific chromatogram. Fourier transform infrared spectrometry（FTIR） was used to study the changes in the chemical composition and content changes of the three samples and to establish their respective FTIR specific chromatogram， while combining hierarchical cluster analysis（HCA）， principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） to find the common and differential characteristics， in order to explore the quantity-quality transfer relationship in the preparation process of standard decoction of Haliotidis Concha（H. discus hannai）. ResultsThe CaCO₃ contents of the 20 batches of medicinal materials， decoction pieces and standard decoction of Haliotidis Concha（H. discus hannai） were 93.87%-98.95%， 96.02%-99.97% and 38.29%-51.96%， respectively， and the extract yield of standard decoction was 1.71%-2.37%， and the CaCO₃ transfer rate of decoction pieces-standard decoction was 0.68%-1.27%. XRF results showed that the elemental species and their relative contents contained in Haliotidis Concha and its calcined products had a high degree of similarity， and although there was no obvious difference in the elemental species contained in decoction pieces and standard decoction， the difference in the relative contents was obvious， which was mainly reflected in the decrease of the relative content of element Ca and the increase of the relative content of element Na. XRD results showed that Haliotidis Concha mainly contained CaCO₃ of aragonite and calcite， while calcined Haliotidis Concha only contained CaCO₃ of calcite， and standard decoction mainly contained CaCO₃ of calcite and Na₂CO₃ of natrite. FTIR results showed that there were internal vibrations of O-H， C-H， C=O， HCO₃^- and CO₃^2- groups in Haliotidis Concha， while O-H， HCO₃^- and CO₃^2- groups existed in the calcined products and standard decoction. ConclusionThe changes of Haliotidis Concha and calcined Haliotidis Concha are mainly the increase of CaCO₃ content， the transformation of CaCO₃ aragonite crystal form to calcite crystal form and the absence of organic components after calcination， and the changes of calcined products and standard decoction are mainly the decrease of CaCO₃ content and the increase of Na₂CO₃ relative content. The method established in the study is applicable to the quality control of the shellfish medicines-decoction pieces- standard decoction， which provides a new idea for the study of quality control of dispensing granules of shellfish medicines.

ObjectiveTo explore the quantity-quality transfer process of medicinal materials， decoction pieces and standard decoction of Haliotidis Concha（Haliotis discus hannai） by analyzing the physical phase and compositional changes， so as to provide references for the effective control of its quality. MethodsA total of 20 batches of Haliotidis Concha（H. discus hannai） from different habitats were collected and prepared into corresponding calcined products and standard decoction， and the content of CaCO₃ of the three samples were determined and the extract yield and transfer rate of CaCO₃ were calculated. The changes in elemental composition and their relative contents were investigated by X-ray fluorescence spectrometry（XRF）， X-ray diffraction（XRD） was used to study the changes in the phase compositions of the three samples and to establish their respective XRD specific chromatogram. Fourier transform infrared spectrometry（FTIR） was used to study the changes in the chemical composition and content changes of the three samples and to establish their respective FTIR specific chromatogram， while combining hierarchical cluster analysis（HCA）， principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） to find the common and differential characteristics， in order to explore the quantity-quality transfer relationship in the preparation process of standard decoction of Haliotidis Concha（H. discus hannai）. ResultsThe CaCO₃ contents of the 20 batches of medicinal materials， decoction pieces and standard decoction of Haliotidis Concha（H. discus hannai） were 93.87%-98.95%， 96.02%-99.97% and 38.29%-51.96%， respectively， and the extract yield of standard decoction was 1.71%-2.37%， and the CaCO₃ transfer rate of decoction pieces-standard decoction was 0.68%-1.27%. XRF results showed that the elemental species and their relative contents contained in Haliotidis Concha and its calcined products had a high degree of similarity， and although there was no obvious difference in the elemental species contained in decoction pieces and standard decoction， the difference in the relative contents was obvious， which was mainly reflected in the decrease of the relative content of element Ca and the increase of the relative content of element Na. XRD results showed that Haliotidis Concha mainly contained CaCO₃ of aragonite and calcite， while calcined Haliotidis Concha only contained CaCO₃ of calcite， and standard decoction mainly contained CaCO₃ of calcite and Na₂CO₃ of natrite. FTIR results showed that there were internal vibrations of O-H， C-H， C=O， HCO₃^- and CO₃^2- groups in Haliotidis Concha， while O-H， HCO₃^- and CO₃^2- groups existed in the calcined products and standard decoction. ConclusionThe changes of Haliotidis Concha and calcined Haliotidis Concha are mainly the increase of CaCO₃ content， the transformation of CaCO₃ aragonite crystal form to calcite crystal form and the absence of organic components after calcination， and the changes of calcined products and standard decoction are mainly the decrease of CaCO₃ content and the increase of Na₂CO₃ relative content. The method established in the study is applicable to the quality control of the shellfish medicines-decoction pieces- standard decoction， which provides a new idea for the study of quality control of dispensing granules of shellfish medicines.

Objective:To investigate the clinicopathological characteristics, molecular features, differential diagnosis and prognosis of renal cell carcinoma (RCC) with TFEB gene amplification.Methods:A total of 113 cases of unclassified RCCs and RCCs with TFEB positive expression were collected from the Affiliated Hospital of Qingdao University and Navy 971 Hospital from January 2010 to December 2024. Eight cases of RCCs with TFEB amplification were identified using tissue microarrays, immunohistochemistry, and fluorescence in situ hybridization (FISH) techniques. The clinicopathological data and prognosis of the 8 cases were summarized, and relevant literature was reviewed.Results:Among the 8 cases, there were 5 males and 3 females. The average age was 63.4 (54, 77) year and the median age was 63.5 (59.0, 65.5) year. Seven cases were detected through physical examination, and 1 case presented with initial symptoms of metastasis to bones and lungs. The cohort included 1 biopsy specimen and 7 surgical resection specimens. The tumor diameters ranged from 2.5 to 15.0 cm. The cut surfaces of 5 cases were grayish-yellow or grayish-red, and 2 cases exhibited a colorful appearance, among which 3 cases involved renal sinus and 1 case showed invasion of the perirenal fat tissue. Microscopically, 4 cases were composed of clear cells arranged in solid sheets or acinar structures, along with varying numbers of eosinophilic cells. Two cases exhibited the morphology of high-grade eosinophilic RCC, and 1 case presented biphasic morphology with diffuse polygonal eosinophilic tumor cells and dense small cell components. The remaining 1 case exhibited the morphology of clear cell RCC. According to the WHO/ISUP nuclear grading system, 6 cases were Grade 3 and 2 cases were Grade 2. Multifocal necrosis was observed in 4 cases. In 4 surgical specimens, the tumor tissue invaded the renal parenchyma, with 2 cases showing nodular infiltration to surrounding tissues and 1 case with intravascular tumor thrombus. Immunohistochemical results showed varying degrees of TFEB nuclear positivity in 6 cases (6/8). Melanocytic markers such as Melan A (5/8) and HMB45 (3/8) were expressed at varying degrees. Cathepsin K (6/8), GPNMB (6/8), P504s (7/8) and CD10 (7/8) were positively expressed in most cases. FISH results revealed high-copy amplification of TFEB gene in 4 cases (partially showing clustered amplification) and low-copy amplification in 4 cases. During the follow-up period of 3 to 64 months of the 8 cases, 3 cases metastasized and 2 cases died of disease (both with high-copy TFEB gene amplification).Conclusions:RCC with TFEB gene amplification is rare and exhibits diverse morphological features. A common morphological characteristic of this type of tumor is a mixture of sheet-like clear cells and high nuclear grade eosinophilic cells. Combined immunohistochemical staining for TFEB, melanocytic markers, and GPNMB is helpful for the diagnosis of the tumor, and FISH detection of TFEB gene amplification is the most definitive method in diagnosing this tumor. RCC with TFEB gene amplification usually presents with strong aggressiveness and poor prognosis. Combining surgical resection with immunotherapy or VEGFR-targeted drugs might have therapeutic effects on the tumor.

Objective:To design an auxiliary device based on embedded microcontroller system for venipuncture,which can adjust posture,so as to resolve the problem that occurs failure in puncture due to insufficient exposure of the puncture site in various scenarios.Methods:The device consisted of a support component,an air ring,a component with lifting and angle adjustment,and a pedestal.By advanced embedded microcontroller technique,it can precisely regulate the posture of the support structure of patient's limbs,and fully expose the targeted puncture site,and create more favorable conditions for nurses in performing punctures.A total of 2,482 patients who underwent blood collection at emergency department of the 305th Hospital of the People's Liberation Army from September to October 2024 were selected.The 1,204 patients were enrolled in September were divided into control group(without using the auxiliary device for venipuncture),and the 1,278 patients were enrolled in October were divided into observation group(using the auxiliary device for venipuncture).The puncture's one-time success rates of junior nurses(experience≤3 years)for both groups were compared.Each group respectively selected 150 patients by using the random number table method to conduct investigate,and satisfaction scores for success rate of puncture,and comfort degree of puncture for position,as well as the pain,process and efficiency,were investigated Results:The puncture's one-time success rates of junior nurses for the patients of control and observation groups were respectively 85.05%and 89.36%,with a statistically significant difference(x2=10.35,P<0.05).The satisfaction scores of patients in the observation group were significantly higher than those in the control group,with a statistically significant difference(t=-5.529,P<0.05).Conclusion:This device is simple and convenient in operation,and has favorable stability.It is beneficial to adjust position and exposure puncture site for patients who undergo peripheral venipuncture.It can improve puncture's success rate and patients'satisfaction.

BACKGROUND:The use of 3D-printed patient-specific instruments in opening wedge high tibial osteotomy has advantages such as shorter operative time,fewer fluoroscopic exposures,and higher correction accuracy.However,previous studies have reported issues such as significant damage to surrounding soft tissues and improper fixation of the plates. OBJECTIVE:To investigate the clinical efficacy of using 3D-printed patient-specific instruments combined with customized locking plate in opening wedge high tibial osteotomy for the treatment of knee osteoarthritis. METHODS:A total of 20 patients diagnosed with knee osteoarthritis were divided into the 3D group(n=10)and the conventional group(n=10)according to surgical methods.The 3D group underwent opening wedge high tibial osteotomy using 3D-printed patient-specific instruments combined with customized locking plate,while the conventional group underwent opening wedge high tibial osteotomy using conventional methods.The operative time,fluoroscopic exposures,incision length,pre-and postoperative hip-knee-ankle angle,medial proximal tibial angle,posterior tibial slope,the difference between the planned and actual correction angle,preoperative and 1,3,6 months postoperative knee range of motion and Lysholm score,and incidence of complications were analyzed and compared between the two groups. RESULTS AND CONCLUSION:(1)The operative time and fluoroscopic exposures were significantly shorter in the 3D group compared to the conventional group,with a statistically significant difference(P<0.001).(2)Both groups showed a significant improvement in postoperative hip-knee-ankle angle and medial proximal tibial angle compared to preoperative values,with a statistically significant difference(P<0.001),while there was no significant change in posterior tibial slope.In the 3D group,the postoperative hip-knee-ankle angle,medial proximal tibial angle,and posterior tibial slope differed from their respective preoperative planned values by(-0.22±0.72)°,(-0.20±0.73)°,and(0.23±0.37)°,but the differences were not statistically significant.The difference between the planned and actual correction angle of 3D group was significantly smaller than that of conventional group(P<0.05).(3)Both groups showed a gradual increase in knee range of motion and Lysholm scores after surgery(P<0.001).Compared to the conventional group,the 3D group had superior knee range of motion at 1 and 3 months postoperatively,as well as a higher Lysholm score at 1 month postoperatively,with statistically significant differences(P<0.05).There were no statistically significant differences in Lysholm score at 3 months and knee range of motion and Lysholm score at 6 months between the two groups(P>0.05).(4)Complications occurred in neither groups.(5)The above results indicate that both 3D-printed patient-specific instruments combined with customized locking plate and conventional methods have good clinical efficacy.However,the former has a shorter operative time,fewer fluoroscopic exposures,and faster postoperative recovery of knee joint function.Additionally,3D-printed patient-specific instruments can achieve preoperative planning accurately.