Objective To investigate the effects of exosomes (Exo) derived from high glucose-stimulated glomerular mesangial cells (GMC) on the kidneys of C57BL/6 mice and the intervention mechanism of Tongluo Yishen Formula (TLYSF). Methods The rat GMC were divided into a normal glucose group (NG, with 5.6 mmol/L glucose) and a high glucose group (HG, with 30 mmol/L glucose). After 24 hours of culture, the supernatant was collected, and exosomes were extracted using the ultracentrifugation method. The exosomes were then identified by transmission electron microscopy and Western blot analysis. Male C57BL/6 mice were divided into three groups: NO-Exo group, NG-Exo group, and HG-Exo group. These groups were respectively administered tail vein injections of PBS buffer, exosomes derived from GMC cultured in normal glucose, and exosomes derived from GMC cultured in high glucose, three times a week for a total of 8 weeks. After 8 weeks, the mice in the HG-Exo group were randomly divided into three subgroups: the HG-Exo group [gavaged with saline], the HG-Exo+TLYSF group [gavaged with TLYSF at 34.32 g/(kg.d)], and the HG-Exo + VAL group [gavaged with valsartan suspension at 10.4 mg/(kg.d)], and the intervention lasted for 4 weeks. Urinary microalbumin (mALb), urinary N-acetyl-β-D-aminoglucosidase (NAG), glycated hemoglobin (HbA1c), serum creatinine (Scr) and urea nitrogen (BUN) were detected. Transmission electron microscopy was used to observe the ultrastructure of renal tissues. TUNEL was used to detect the DNA damage of renal tissue cells. Immunofluorescence was used to detect the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) and wilms tumor 1(WT-1). RT-PCR was used to detect the mRNA levels of NLRP3, cysteinyl aspartate-specific proteinase 1 (caspase-1), interleukin-1 beta (IL-1β), miR-200c-3p and miR-148a-3p. Western Blot was employed to detect the protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 and IL-1β. Results Compared with the NG-Exo group, mice in the HG-Exo group exhibited significantly increased levels of mALb, urinary NAG, Scr and BUN. Transmission electron microscopy revealed ruptured podocyte membranes and swollen mitochondria. The positive rate of cells stained by the TUNEL increased, with elevated optical density of NLRP3 and decreased optical density of WT-1. Additionally, there was a significant increase in the level of NLRP3, caspase-1, IL-1β mRNA, as well as miR-200c-3p and miR-148a-3p. The protein expression of NLRP3, ASC, caspase-1, and IL-1β also increased. Compared with HG-Exo group, mice in the HG-Exo+TLYSF group showed decreased levels of mALb, urinary NAG, Scr, and BUN. The podocyte membranes were relatively intact, and mitochondrial damage was alleviated. The positive rate of cells stained by the TUNEL decreased, along with a reduction in the optical density of NLRP3 and an increase in the optical density of WT-1. Furthermore, the mRNA expression levels of NLRP3, caspase-1, IL-1β, miR-200c-3p, and miR-148a-3p were all downregulated to varying degrees. The protein expression levels of NLRP3, ASC, caspase-1, and IL-1β also decreased. Conclusion Exosomes derived from GMC stimulated by high glucose can damage the kidneys of mice and induce podocyte pyroptosis. TLYSF may ameliorate podocyte pyroptosis by downregulating the expression of exosomal miR-200c-3p and miR-148a-3p and inhibiting the activation of the NLRP3/ASC/caspase-1 pathway.
Animals ; Exosomes/ultrastructure* ; Glucose/pharmacology* ; Male ; Podocytes/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Mice, Inbred C57BL ; Mice ; Mesangial Cells/metabolism* ; Pyroptosis/drug effects* ; Rats ; MicroRNAs/genetics*

Objective:To explore the influence of voice training combined with active breathing and circulation techniques on voice recovery following vocal cord polyp surgery. Methods:A total of 110 patients who underwent vocal cord polyp surgery at our hospital from May 2022 to November 2023 were selected and randomly divided into a control group （n=55） and a combination group （n=55） using a random number table method. During the recovery period, both groups received dietary control and aerosol treatment. The control group participated in voice training, while the combination group received active breathing and circulation techniques in addition to voice training for 2 months. Morphological changes, voice acoustic indicators （Shimmer, Jitter, Maximum Phonation Time[MPT]）, and the Voice Handicap Index （VHI） were compared between the two groups, and clinical efficacy was evaluated. Results:The combination group demonstrated higher clinical efficacy after training compared to the control group, with a statistically significant difference （P<0.05）. The proportion of incomplete closure, abnormal mucosal wave, and supraglottic compensation decreased in both groups after training （P<0.05）. However, there was no significant difference in the proportions of incomplete closure and abnormal mucosal wave between the two groups （P>0.05）. Notably, the proportion of patients with supraglottic compensation in the combination group was lower than in the control group （P<0.05）. After training, the Shimmer and Jitter values decreased in both groups, with the combination group exhibiting lower values （P<0.05）. Conversely, the MPT values increased in both groups, again with higher values in the combination group （P<0.05）. Additionally, after training, the functional, physiological, and emotional scores of the VHI decreased in both groups, with the scores in the combination group lower than those in the control group, demonstrating statistical significance （P<0.05）. Conclusion:Voice training combined with active breathing and circulation techniques has a beneficial effect on recovery following vocal cord polyp surgery. This combined approach significantly improves vocal cord morphology and acoustic indices, alleviates voice disorders, and enhances overall voice recovery.
Humans ; Vocal Cords/surgery* ; Polyps/surgery* ; Voice Training ; Male ; Female ; Voice Quality ; Laryngeal Diseases/surgery* ; Voice ; Middle Aged ; Adult ; Respiration

OBJECTIVE To investigate the relationship between azimuth direction,angular intervals,and the accuracy of azimuthal sound source localization.METHODS Fifteen young subjects with normal hearing were tested using nine azimuth settings.The test results were presented as root mean square error and percentage confusion.RESULTS The confusion rate under high-frequency narrowband noise was significantly higher than that under broadband noise and three-syllable words.In the frontal direction,statistically significant differences were observed between the 20° and 10° intervals,as well as between the 20° and 15° intervals(P<0.05),but no significant difference was found between the 10° and 15° intervals(P>0.05).In the lateral and rear directions,statistically significant differences were found between the 30° and 15° intervals,as well as between the 30° and 20° intervals(P<0.05),but no significant difference was found between the 15° and 20° intervals(P>0.05).Statistically significant differences were observed between the frontal direction and both the lateral and rear directions(P<0.05),but no significant difference was found between the lateral and rear directions(P>0.05).CONCLUSION Using stimuli that contain broader bandwidth cues can more accurately reflect the subject's horizontal localization ability.For source azimuth identification tests using broadband noise and three-syllable words,it is recommended to use a 15° interval in the frontal direction,and a 20° interval in the lateral and rear directions.The frontal and lateral directions can be preferred for testing.

Objective The high post-surgery recurrence rate of endometriosis(EMs)has emerged as a challenge in the long-term manaagement of the condition.This study is aimed at investigating the mechanisms of Neiyiting(NYT)decoction in preventing postoperative recurrence of EMs.Methods An animal model of EMs postoperative recurrence and a model of endometrial stromal cells(hEM15A)cocultured with macrophages(RAW 264.7 cell line)were established for both in vivo and in vitro experiments.An autotransplantation method was used to establish a rat model of EMs.The rats were divided into 4 groups(6 rats per group)and received the corresponding treatments:a Model group receiving distilled water,a Gestrinone group receiving gestrinone at 0.325 mg/kg,a low-dose NYT(NYT-L)group receiving NYT decoction at 5.04 g/(kg-d),and a high-dose NYT(NYT-H)group receiving NYT decoction at 10.08 g/(kg-d).The treatment was administered for 3 weeks via intragastric gavage.In addition,6 SD rats were randomly selected for the control group(Control group),and were given distilled water for 3 weeks via intragastric gavage.The sizes and pathological changes of recurrent lesions in EMs rats were observed.Immunohistochemistry and qRT-PCR were performed to assess the expression of M1 macrophage marker CD86 protein and mRNA in vivo.Additionally,immunohistochemistry and qRT-PCR were used to assess the expression of indicator proteins related to the triggering receptor expressed on myeloid cells 1(TREM1)/Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)signaling pathway and mRNA.The proliferation of hEM15A cells in the coculture experiment was observed.Flow cytometry was performed to determine the polarization of RAW264.7 macrophages,and qRT-PCR was used to determine the expression levels of inducible nitric oxide synthase(iNOS)and interleukin 1β(IL-1β)mRNA.Western blot was performed to determine the expression of signaling pathway-related indicator proteins in vitro.ELISA was performed to determine the levels of inflammatory factors in vitro.Results Compared with the Model group,the volume of recurrent lesions in the NYT-H group was reduced(P＜0.01).Findings from the macrophage M1 polarization assessment showed that the expression levels of CD86 protein and mRNA in the recurrent lesions of the Model group were higher than those in the control group(P＜0.01).The expression levels of CD86 protein and mRNA in the recurrent lesions of the NYT-H group were lower than those of the Model group(P＜0.01).In addition,the RAW 264.7 cell experiment further verified that NYT decoction could reduce the number of CD86-positive macrophages induced by plasmids overexpressing TREM1 and reduce the expression of IL-1β and iNOS mRNA(P＜0.01).The results of the hEM15A cell proliferation assay showed that NYT decoction down-regulated KI-67 protein expression in hEM15A cells induced by macrophage M1 polarization(P＜0.01).The results of TREM1/TLR4/NF-κB signaling pathway showed that the protein and mRNA expression levels of TREM1,TLR4,and NF-κB in the recurrent lesions of the Model group were higher than those of the control group(P＜0.01).Compared with those in the Model group,the protein and mRNA expression levels of TREM1,TLR4,and NF-κB in the recurrent lesions of the NYT-H group were lower(P＜0.01).In addition,the coculture experiment of RAW264.7 and hEM15A cells further confirmed that NYT decoction reduced the expression of TREM1,TLR4,and P-P65 proteins(P＜0.01).Conclusion NYT decoction can inhibit macrophage M1 polarization through the TREM1/TLR4/NF-κB signaling pathway,improve the inflammation level,and inhibit the formation of ectopic endometrial lesions,thereby preventing postoperative recurrence of EMs.

Objective To observe the characteristics of gut microbiota in patients with type 2 diabetes mellitus(T2DM)with different traditional Chinese medicine(TCM)syndrome types,and to further explore the key microbial communities and functional differences affecting syndrome differentiation.Methods A total of 45 patients who visited the Department of Geriatrics,Hunan Provincial Hospital of Integrated Traditional Chinese and Western Medicine in 2023 were enrolled.These included 15 T2DM patients with qi-yin deficiency and blood stasis syndrome(Group A),15 T2DM patients with qi-yin deficiency syndrome(Group B),and 15 non-diabetic patients from the same period(Group C).Fecal samples were collected,and 16S rRNA sequencing and analysis were performed.Results 1)A total of 1 564 operational taxonomic units(OTUs)were obtained from the three groups of patients,with 224,127,and 351 unique OTUs identified in Groups A,B and C,respectively.2)Both α-and β-diversity analyses indicated differences among the gut microbiota of the three groups.For instance,in the α-diversity analysis,the Sobs index showed significant inter-group differences(P＜0.01).Group A(264.00±88.84)was significantly higher than Group B(145.90±87.0)(P＜0.01),while Group B was significantly lower than Group C(229.7±112.4)(P＜0.05).In the β-diversity analysis,the principal coordinate analysis(PCoA)indicated a clear separation among groups(R=0.1610,P＜0.01).The R values in the Anosim/Adonis analysis ranged from 0.144 to 0.196,and the R2 values ranged from 0.067 to 0.083,all indicating differences in inter-group comparisons(P＜0.01).3)At the phylum level,Firmicutes,Actinobacteriota,and Bacteroidota were predominant in all groups.Among them,Bacteroidota exhibited significant inter-group differences(P＜0.05),with its abundance in Group A being significantly higher than that in Group B(P＜0.01).4)Analysis of differences in microbiota composition,combined with linear discriminant analysis effect size(LEfSe)and Random Forest analysis,revealed that,at the genus level,the microbiota biomarkers between Group A and Group B were Parabacteroides,Bacteroides,g_unclassified_f_Lachnospiraceae,Roseburia,and Aspergillus,those between Group B and Group C were Erysipelotrichaceae_UCG-003 and Ruminococcus,and those between Group A and Group C were Parabacteroides,Anaerotruncus,and Oscillibacter.The results were validated by receiver operating characteristic(ROC)curve analysis,which suggested that the microbiota biomarkers between Group A and Group B(AUC=0.91;95％CI,0.80-1.00),Group B and Group C(AUC=0.84;95％CI,0.69-0.99),Group A and Group C(AUC=0.87;95％CI,0.75-0.99)had good diagnostic efficacy.5)The study identified 116 major pathways with inter-group differences through Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.For example,the enrichment degree of ABC transporter pathway in Group A(2.58±0.36)was significantly lower than those in Group B(2.90±0.48)and Group C(3.11±0.66)(P＜0.05).These pathways were associated with metabolism and environmental information processing.g.Conclusion The differences in the gut microbiota characteristics and functions among patients with specific TCM syndromes of T2DM may provide references for TCM syndrome differentiation and therapeutic mechanisms.

Objective To investigate the expressions of glycolysis-related factors and changes in Notch1 signaling in endometrial tissues of adenomyosis(AM)and Ishikawa cells to explore the pathogenesis of AM.Methods Eutopic endometrial tissues were collected from 8 patients with AM and 8 patients with uterine fibroids matched for clinical characteristics(control group).The expressions of Notch1 signaling proteins and glycolysis-related factors in the collected tissues were detected using qRT-PCR and Western blotting,and the levels of glucose and lactic acid were determined.An Ishikawa cell model with lentivirus-mediated stable Notch1 overexpression was established for assessing cell survival rate with CCK-8 assay,cell migration and invasion abilities with Transwell migration and invasion assays,and glycolytic capacity by determining the extracellular acidification rate.Results Compared with those in the control group,the endometrial tissues in AM group showed significantly increased expression level of carbohydrate antigen 125(CA125),increased mRNA expression levels of Notch1,HK2 and PDHA and protein expressions of Notch1,GLUT1,HK2,PKM and PDHA,lowered glucose level and increased lactate level.The Ishikawa cell models with stable Notch1 overexpression exhibited significantly increased cell survival rate with attenuated cell migration and invasion abilities and decreased glycolysis capacity and reserve.Conclusion The Notch1 signaling pathway participates in the pathogenesis of AM possibly by regulating the proliferation,migration,invasion and glycolysis of endometrial cells.

The methodological framework for interventional clinical research of Chinese medicine （CM） under the research mode of syndrome dominating disease provides a set of technical principles and methods to design， evaluate， and implement of this kind. It consists of three main parts including general principles， research points and key design elements， with a total of 25 items. This methodological framework proposes implementing requirements and recommendations in a variety of aspects， including basic norms to be followed in relevant studies， perspectives for selecting research topics， as well as the technological details on study population （P）， intervention （I） and comparison（C）， outcome measurement （O）， time frame （T） of treatment and follow-up， sample orientation （prospective versus retrospective）， study design （S） format and type. To provide practical guidance for future studies， this article clearly explains each items of the methodological framework through some supportive cases.

Objective To investigate the expressions of glycolysis-related factors and changes in Notch1 signaling in endometrial tissues of adenomyosis(AM)and Ishikawa cells to explore the pathogenesis of AM.Methods Eutopic endometrial tissues were collected from 8 patients with AM and 8 patients with uterine fibroids matched for clinical characteristics(control group).The expressions of Notch1 signaling proteins and glycolysis-related factors in the collected tissues were detected using qRT-PCR and Western blotting,and the levels of glucose and lactic acid were determined.An Ishikawa cell model with lentivirus-mediated stable Notch1 overexpression was established for assessing cell survival rate with CCK-8 assay,cell migration and invasion abilities with Transwell migration and invasion assays,and glycolytic capacity by determining the extracellular acidification rate.Results Compared with those in the control group,the endometrial tissues in AM group showed significantly increased expression level of carbohydrate antigen 125(CA125),increased mRNA expression levels of Notch1,HK2 and PDHA and protein expressions of Notch1,GLUT1,HK2,PKM and PDHA,lowered glucose level and increased lactate level.The Ishikawa cell models with stable Notch1 overexpression exhibited significantly increased cell survival rate with attenuated cell migration and invasion abilities and decreased glycolysis capacity and reserve.Conclusion The Notch1 signaling pathway participates in the pathogenesis of AM possibly by regulating the proliferation,migration,invasion and glycolysis of endometrial cells.

Objective:To investigate the feasibility of a three-dimensional no new U-Net (3D nnU-Net) deep learning (DL) network for the automatic segmentation of colorectal cancer (CRC) based on abdominal CT images.Methods:This was a cross-sectional study. From January 2018 to May 2023, a total of 2180 primary CRC patients, confirmed by pathology at the Guangdong Provincial Hospital of Traditional Chinese Medicine (center 1, n=777), Nanfang Hospital, Southern Medical University (center 2, n=732), and Sun Yat-sen Memorial Hospital (center 3, n=671), were enrolled in this retrospective study. The baseline abdominal CT examination of each patient was conducted using CT equipment from 7 different models across 4 vendors, at the 3 centers, encompassing both the arterial phase (AP) and venous phase (VP). Two radiologists manually delineated the volume of interest to circumscribe the entire tumors in dual-enhanced phase CT images. The CT data of CRC patients from center 1 and center 3 were merged and divided into a training set ( n=1 159) and a validation set ( n=289) using a weighted random method with a ratio of 4∶1. The patients from center 2 were used as an independent external test set ( n=732). The 3D nnU-Net segmentation model was trained and tested. Using manually annotated label data as the benchmark, segmentation performance of the model was evaluated based on different phases and tumor locations. The segmentation coverage rate (SCR), Dice similarity coefficient (DSC), recall (REC), precision (PRE), F1-score, and 95% Hausdorff distance (HD 95) were calculated. The mean manual segmentation time and the mean automatic time were compared using independent samples t-test. Results:In the independent external test set, the performance of the 3D nnU-Net model based on the AP CT images was superior to that based on the VP CT images. On the AP images, the SCR, DSC, REC, PRE, F1-score, and HD 95 were 0.865, 0.714, 0.716, 0.736, 0.714, and 27.228, respectively; on the VP images, they were 0.834, 0.679, 0.710, 0.675, 0.679, and 29.358, respectively. The model achieved the best performance on right-sided colon cancer, with SCR, DSC, REC, PRE, F1-score, and HD95 on the AP CT images at 0.901, 0.775, 0.780, 0.787, 0.775, and 21.793, respectively. Next were left-sided colon cancer and rectal cancer, while the segmentation performance for transverse colon cancer was the worst (SCR, DSC, REC, PRE, F1-score, and HD 95 were 0.731, 0.631, 0.641, 0.630, 0.631 and 38.721, respectively). The automatic segmentation time on a single phase was (1.0±0.3) min, while the manual segmentation time was (17.5±6.0) min ( t=128.24, P<0.001). Conclusions:After training and validating on a dataset from multiple centers with various CT scanner vendors, the 3D nnU-Net DL model demonstrates the capability to automatically segment CRC based on abdominal CT images, while also showcasing commendable robustness and generalization ability.