1.Action Mechanism of Huamoyan Granules in Treatment of Knee Osteoarthritis Based on TRPV1/p38 MAPK Pathway
Jin ZHANG ; Lili YANG ; Canwen ZHENG ; Jing KANG ; Yanlei MA ; Yue SHI ; Lei LI ; Hongxu MENG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):79-89
ObjectiveThis paper aims to observe the protective effect of Huamoyan granules on knee osteoarthritis (KOA) and explore whether its protective effect is oriented toward an anti-inflammatory direction by regulation of macrophage polarization, which can effectively inhibit the progression of pathological inflammatory response, reduce the release of inflammatory pain mediators, and downregulate the protein expression level of transient receptor potential vanilloid 1 (TRPV1), so as to provide experimental evidence for its clinical application and investigate its action mechanism. MethodsAfter adaptive feeding, Sprague-Dawley (SD) rats were randomly divided into six groups: sham group, model group, celecoxib group, and high, medium, and low-dose synovitis granule groups (9.6, 4.8, 2.4 g·kg-1). The administration dose of celecoxib capsules was 20 mg·kg-1. There were 10 rats in the sham group and 12 rats in the model group and each administration group. A KOA animal model was established by means of intra-articular injection of sodium iodoacetate into the knee joint. From the 10th day of the experiment, each administration group was given intragastric administration at a dose of 10 mL·kg-1 for 4 weeks. General conditions of rats in each group were assessed daily. The pressure pain threshold (PPT) to mechanical stimulation and joint diameter were recorded. X-ray examination was performed on the right knee joints of rats for imaging analysis. Enzyme linked immunosorbent assay (ELISA) was performed to detect the tumor necrosis factor-α (TNF-α), serum interleukin-1β (IL-1β), and other pro-inflammatory cytokines in rat serum samples, as well as the expression levels of neurogenic inflammatory mediators such as nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP). Histopathological changes in the knee joint synovial tissues were examined by hematoxylineosin (HE) staining. Safranin O-fast green staining was performed to observe and evaluate the degree of knee cartilage lesions. Western blot was employed to quantitatively analyze TRPV1, p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated (p)-p38 MAPK in rat knee synovial tissues. Immunofluorescence (IF) was used to measure and assess M1/M2 macrophage polarization. ResultsCompared with those in the sham group, the circumference and joint diameter of the right knee were markedly enlarged in the model group (P<0.01), while PPTs of rats showed a significant reduction (P<0.01). The contents of IL-1β, TNF-α, CGRP, and NGF in rats' serum were significantly elevated (P<0.01), and the synovial Krenn score was increased (P<0.01). The Mankin score of cartilage tissue was increased (P<0.01), and the protein expressions of TRPV1 and p-p38 MAPK/p38 MAPK were significantly upregulated (P<0.01). The experimental intervention significantly reduced the proportion of pro-inflammatory M1 macrophages in the total macrophage population (P<0.01), and the percentage of M2 macrophages was decreased (P<0.01). The M1/M2 macrophage ratio was significantly elevated (P<0.01). Knee joint diameters of all dose groups of Huamoyan granules and the celecoxib group were reduced (P<0.01) compared with those of the model group, and the PPT recovery speeds in the high and medium-dose groups of Huamoyan granules were more obvious (P<0.05). The contents of IL-1β, CGRP, and NGF in the rats' serum in all administration groups were significantly reduced (P<0.05, P<0.01), and the content of TNF-α in rats' serum was significantly reduced (P<0.01). All dose groups of Huamoyan granules demonstrated significant reductions in both synovial Krenn score (P<0.05, P<0.01) and protein expression of TRPV1 and p-p38 MAPK/p38 MAPK in rats' synovial tissues (P<0.01). The percentage of M1 macrophages in the synovial tissues of the celecoxib group and all dose groups of Huamoyan granules was decreased (P<0.01). The percentage of M2 macrophages was increased (P<0.05), and the M1/M2 ratio was decreased (P<0.01). ConclusionHuamoyan granules can alleviate the inflammatory response of KOA, reduce the release of inflammatory pain mediators, and downregulate TRPV1 protein expression by regulating macrophage polarization. Its mechanism may be related to the TRPV1/p38 MAPK signaling pathway, thereby achieving the effect of improving peripheral pain hypersensitivity in KOA.
2.Exploring Mechanism of Xiaojinwan-containing Serum in Inhibiting Breast Cancer Cells Based on Wnt/β-catenin Signaling Pathway
Hongdi TANG ; Xiaobo JIN ; Chen GAO ; Hongkai LI ; Zhiming LI ; Lu YAO ; Fengli SONG ; Ning KANG ; Wenwen WANG ; Ying SUN ; Zhibin WANG ; Tong LI ; Yingli ZHU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(17):13-22
ObjectiveTo investigate the role and underlying mechanism of the Wnt/β-catenin signaling pathway in the process of Xiaojinwan (XJW)-containing serum interfering with endothelial-mesenchymal transition (EMT) in human breast cancer MDA-MB-231 cells. MethodsXJW-containing serum was prepared by intragastric administration of XJW (4 g·kg-1) to SD rats. MDA-MB-231 cells were cultured in vitro. β-Catenin overexpression and knockdown plasmids were constructed and transfected into MDA-MB-231 cells. The optimal XJW-containing serum (4 g·kg-1) according to previous studies was used for β-catenin intervention and overexpression experiments. The groups for intervention/overexpression experiments included intervention/overexpression blank group, β-catenin intervention/overexpression group, intervention/overexpression blank + XJW group, and β-catenin intervention/overexpression + XJW group. Cell morphology was observed under a microscope. Flow cytometry was used to detect and record apoptosis rates. Western blot was performed to measure apoptosis-related proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and active-Caspase-3] and EMT-related proteins (E-cadherin, N-cadherin, and vimentin). Real-time polymerase chain reaction (Real-time PCR) was used to detect β-catenin mRNA expression levels in different groups. ResultsIntervention experiment results showed that compared with the intervention blank group, the β-catenin intervention group and the intervention blank + XJW group showed increased apoptosis rates (P<0.05, P<0.01), elevated protein levels of Bax, active Caspase-3, and E-cadherin (P<0.05, P<0.01), and decreased protein levels of Bcl-2 and Vimentin (P<0.05, P<0.01). β-catenin mRNA expression was reduced in the β-catenin intervention group (P<0.05, 0.63 ± 0.15) and the intervention blank + XJW group (P<0.05, 0.79 ± 0.33). Compared with the β-catenin intervention group, the β-catenin intervention + XJW group exhibited an increased apoptosis rate (P<0.01), elevated Bax and active Caspase-3 protein levels (P<0.05, P<0.01), and decreased Bcl-2, N-cadherin, and Vimentin protein levels (P<0.05). β-catenin mRNA expression was further reduced in the β-catenin intervention + XJW group (P<0.01, 0.22 ± 0.08). Compared with the intervention blank + XJW group, the β-catenin intervention + XJW group showed an increased apoptosis rate (P<0.01), elevated Bax, active Caspase-3, and E-cadherin protein levels (P<0.05, P<0.01), and decreased Bcl-2, N-cadherin, and vimentin protein levels (P<0.05, P<0.01). β-catenin mRNA expression was also reduced (P<0.05). Overexpression experiment results showed that compared with the overexpression blank group, the β-catenin overexpression group exhibited a reduced apoptosis rate (P<0.01), while the overexpression blank + XJW group showed an increased apoptosis rate (P<0.05). In the β-catenin overexpression and β-catenin overexpression + XJW groups, Bax and E-cadherin protein levels decreased (P<0.05, P<0.01), while Bcl-2, N-cadherin, and vimentin levels increased (P<0.05). In the overexpression blank + XJW group, Bax and E-cadherin levels increased (P<0.05, P<0.01), while Bcl-2, N-cadherin, and vimentin levels decreased (P<0.05, P<0.01). β-catenin mRNA expression increased in the β-catenin overexpression group (P<0.01, 7.26 ± 1.03) but decreased in the overexpression blank + XJW group (P<0.05, 0.73 ± 0.14). Compared with the β-catenin overexpression group, the β-catenin overexpression + XJW group showed an increased apoptosis rate (P<0.01), elevated Bax and E-cadherin levels (P<0.05), and decreased N-cadherin and vimentin levels (P<0.05). β-catenin mRNA expression was reduced (P<0.05, 4.65 ± 1.02). Compared with the overexpression blank + XJW group, the β-catenin overexpression + XJW group exhibited a reduced apoptosis rate (P<0.01), decreased Bax and E-cadherin levels (P<0.01), and increased Bcl-2, N-cadherin, and Vimentin levels (P<0.05). β-catenin mRNA expression increased (P<0.01). ConclusionXJW-containing serum inhibits breast cancer cell proliferation by suppressing EMT, which is associated with the regulation of the Wnt/β-catenin pathway.
3.Traditional Chinese Medicine Regulates Immune Escape to Ameliorate Gastrointestinal Tumors: A Review
Pengpeng LIU ; Li KANG ; Chengdan MAO ; Xueling YUAN ; Jin SHU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(17):282-290
Malignant gastrointestinal tumors (GT) mainly include laryngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, bile duct cancer, and colorectal cancer. At present, the morbidity and mortality of GT are high. Tumor immune escape is one of the key reasons for cancer development and treatment failure, and it is also the main direction of current tumor immunotherapy research. Traditional Chinese medicine (TCM) plays a role in treating GT and alleviating systemic symptoms, especially showing potential advantages in regulating the immune escape mechanism. This article elaborates on the immune escape mechanism of GT and systematically reviews that TCM active components or compound prescriptions act on key immune checkpoints such as programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) to reduce the proportion of marrow-derived suppressor cells (MDSCs), promote the polarization of M1 macrophages, activate effector immune cells, enhance CD8+ T cell recruitment and interferon (IFN)-γ secretion, enhance the killing activity of natural killers (NKs), and reduce foxhead box protein P3(FoxP3+)-positive regulatory T (Treg) cell infiltration. TCM active components or compound prescriptions can remodel the tumor microenvironment (TME), such as improving immunosuppressive factors, promoting vascular normalization, and regulating intestinal microbiota. Through the regulation of TME, enhancement of antigen presentation, inhibition of immune checkpoint molecules, regulation of cellular network factors and metabolites, etc., TCM active components or compound prescriptions enhance the immune function, thereby inhibiting the immune escape of GT. This paper summarizes and reflects on the research results of TCM in regulating immune escape to treat GT in recent years, aiming to help transform TCM from empirical medication to precision treatment based on immune regulation, reduce drug resistance and enhance treatment effect.
4.Objective Indicators and Evaluation System for Excellent Shape and High Quality of Gardeniae Fructus
Xiulin YUAN ; Shiwen KANG ; Zhihao LIU ; Ding HUANG ; Sen YE ; Fan LI ; Bocheng WANG ; Yan JIN ; Yuan YUAN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(18):27-35
ObjectiveTo establish an objective evaluation system based on the quality characteristics of Gardeniae Fructus without relying on the origin label according to the theory of excellent shape, high quality, and superior effect of authentic medicinal materials, thus providing a scientific basis for the standardization of Gardeniae Fructus. MethodsDifferent batches of Gardeniae Fructus samples were collected from different producing areas. Through herbal textual research and a review of modern literature, fruit length, maximum fruit diameter, and chroma were selected as quantitative indicators to characterize the traditional appearance traits of Gardeniae Fructus. The above indicators were measured by a vernier caliper and a spectrophotometer. Through classical famous prescription data and literature analysis, total iridoid glycosides, total crocus glycosides, and total flavonoids were selected as the core component indicators related to efficacy, and their content was determined. Finally, through cluster analysis, the threshold ranges of the quantitative indicators of excellent shape and high quality were determined, and the applicability of the established evaluation system was verified by the determination of the indicators for each batch of medicinal materials. ResultsThe excellent shape indicators of Gardeniae Fructus were the fruit length-to-maximum fruit diameter ratio ≤1.86, 0°≤h≤45°, and L*≥ 37.32. The content of total iridoid glycosides, total crocus glycosides, and total flavonoids should be no less than 2.2%, 0.44%, and 0.028%, respectively. The measurement results of different batches of Gardeniae Fructus showed that the evaluation system could effectively distinguish the Gardeniae Fructus samples in line with the traditional cognition of excellent shape and high quality. ConclusionThis study preliminarily constructs an excellent shape and high quality-based quality evaluation system for Gardeniae Fructus, which links appearance traits with chemical indicators and provides a methodological basis for the quality control and scientific grading of this herbal medicine.
5.Construction and preparation of virus-like particle expression vector for yellow fever virus
Miaomiao LYV ; Chunying GAO ; Yunfeng LI ; Zhiying JIN ; Min WAN ; Jie TIAN ; Weina KANG
Journal of Public Health and Preventive Medicine 2026;37(5):12-16
Objective To prepare virus-like particles (VLPs) for yellow fever virus (YFV) using the pACYCDuet-1 single-plasmid dual expression system. Methods Gene fragments including NS1, NS5, and 5' untranslated region (UTR) and 3' UTR fragment of YFV were cloned into the expression plasmid pACYCDuet-1-MS2-TMpac (carrying MS2 coat protein gene) and transformed into E. coli BL21(DE3) for expression. VLPs were observed by TEM. After DNase I/RNase A digestion, RNA was extracted and transcribed into cDNA, which was subjected to fluorescence quantitative PCR detection. Results According to SN/T 3560-2013, double digestion and colony PCR of pACYCDuet-1-MS2-TMpac-YFV both produced expected bands. SDS-PAGE showed a target band at 25-35 kDa. RT-PCR amplified VLPs after enzyme digestion, and TEM revealed VLPs with clear edges and translucent interiors. Thus, the plasmid was successfully constructed, and the phage coat protein packaged YFV genes with nuclease resistance. Conclusion VLPs containing specific gene fragments of yellow fever virus have been successfully prepared. These nuclease-resistant VLPs can serve as safe and stable quality control materials to ensure the reliability of laboratory detection for YFV.
6.Comparison of clinical characteristics between primary bilateral macronodular adrenal hyperplasia and adrenal cortisol-producing adenoma
Bing LI ; Ming-Xiu YANG ; Huai-Jin XU ; Jing-Xuan WANG ; Qing-Zheng WU ; Ya-Jing WANG ; Yi-Jun LI ; Kang CHEN ; Yu CHENG ; Qi NI ; Ya-Qi YIN ; Li ZANG ; Qing-Hua GUO ; Jian-Ming BA ; Wei-Jun GU ; Jing-Tao DOU ; Zhao-Hui LYU ; Yi-Ming MU
Medical Journal of Chinese People's Liberation Army 2025;50(7):779-785
Objective To comparatively analyze the clinical characteristics of primary bilateral macronodular adrenal hyperplasia(PBMAH)and adrenal cortisol-producing Adenoma(CPA),and enhance the understanding of two diseases.Methods The clinical data of 85 PBMAH patients(PBMAH group)and 195 CPA patients(CPA group)diagnosed at Department of Endocrinology,the First Medical Center of Chinese PLA General Hospital,from September 2014 to August 2024 were retrospectively analyzed.The demographic characteristics,comorbidities,biochemical indicators,adrenocorticotropic hormone-cortisol(ACTH-F)levels,and adrenal imaging features and treatment conditions were compared between the two groups.Results(1)General characteristics:Compared with CPA group,PBMAH group had older age at diagnosis and a higher proportion of male patients.(2)Clinical characteristics:Compared with CPA group,PBMAH group had a longer disease duration,a higher proportion of subclinical Cushing's syndrome(CS),and a higher proportion of hypertension,impaired glucose tolerance/diabetes,bone mass reduction or osteoporosis,with higher serum potassium levels,and the differences were statistically significant(P<0.01).(3)Hormone levels:Both PBMAH and CPA groups showed ACTH-F rhythm disorder,significantly increased cortisol levels and suppressed ACTH.Compared with PBMAH group,CPA group had stronger autonomous cortisol secretion ability,manifested by increased midnight serum cortisol(F0:00),16:00 serum cortisol(F16:00),24-hour urinary free cortisol(24 h UFC)levels and lower 8:00 serum ACTH(ACTH8:00)and 16:00 serum ACTH(ACTH16:00)(P<0.01).After low-dose dexamethasone suppression test(LDDST),CPA group showed lower suppression rates of ACTH and cortisol,and higher proportions of paradoxical elevation in serum cortisol and 24 h UFC compared with PBMAH(P<0.01).Conclusions PBMAH has a longer disease course and higher proportions of comorbid metabolic disorders than CPA,mostly manifested as subclinical Cushing's syndrome.CPA has stronger autonomous cortisol secretion ability,with cortisol less likely to be suppressed after LDDST and more obvious paradoxical elevation of cortisol and 24 h UFC.
7.Characteristics analysis of multimodal metabolic disorders in subclinical Cushing's syndrome patients with different cortisol levels
Ya-Jing WANG ; Bing LI ; Huai-Jin XU ; Qi NI ; Ya-Qi YIN ; Yi-Jun LI ; Li ZANG ; Yu CHENG ; Kang CHEN ; Qing-Hua GUO ; Jian-Ming BA ; Wei-Jun GU ; Jing-Tao DOU ; Zhao-Hui LYU ; Yi-Ming MU
Medical Journal of Chinese People's Liberation Army 2025;50(7):793-799
Objective To characterize multimodal metabolic disorders in subclinical Cushing's syndrome(SCS)patients with different cortisol levels,providing a reference for clinical diagnosis and treatment.Methods A retrospective analysis was conducted on the clinical data of 165 SCS patients diagnosed at the First Medical Center of Chinese PLA General Hospital due to adrenal masses from January 2014 to October 2024.Using the serum cortisol levels after the midnight 1 mg dexamethasone suppression test(1 mg DST)as the cut-off point,SCS patients were divided into high-level group(1 mg DST-F>138 nmol/L,n=96)and low-level group(50 nmol/L<1 mg DST-F≤138 nmol/L,n=69).The differences in age,gender,body mass index(BMI),blood pressure,glucolipid metabolism indices,electrolytes,hormone levels,and imaging features of adrenal adenoma(such as CT values)were compared between the two groups.Multivariate linear regression was used to analyze the correlation between CT values and metabolic indices.Results Compared with low-level group,patients in high-level group were younger(54.0±11.3 vs.57.7±10.3,P=0.034),while there were no statistically significant differences in gender ratio or BMI between the two groups(P>0.05).Both groups exhibited decreased adrenocorticotropic hormone(ACTH)levels and disrupted circadian rhythm.Compared with low-level group,high-level group showed significantly higher F0:00 levels[250.00(170.07,422.53)nmol/L vs.110.00(82.74,133.90)nmol/L]and 24-hour urinary free cortisol(24 h UFC)[568.40(377.80,875.45)nmol/24 h vs.369.40(265.40,494.69)nmol/24 h](P<0.001),with no significant differences in serum F8:00,or 1 mg DST ACTH0:00 levels(P>0.05).Except for the fasting C-peptide level in the high-level group being higher than that in low-level group[(2.88±1.01)ng/ml vs.(2.46±0.78)ng/ml,P=0.024],there were no significant differences in blood pressure,blood lipids,glycated hemoglobin(HbA1c),fasting blood glucose,fasting insulin,serum electrolytes,uric acid,and other indices between the two groups(P>0.05).The CT value of adrenal adenoma during contrast-enhanced scanning was higher in high-level group[80.00(17.80,93.00)Hu vs.52.00(35.50,75.00)Hu,P=0.006]compared with low-level group.Multivariate linear regression analysis revealed that diastolic blood pressure was positively correlated with CT values of adrenal adenomas in both plain scanning(β=0.49,95%CI 0.09-0.90)and contrast-enhanced scanning(β=2.08,95%CI 0.76-3.39),while triglyceride levels were negatively correlated with plain scanning CT values(β=-5.77,95%CI-10.88--0.66).Conclusion Patients with SCS at different cortisol levels differ in age,fasting C-peptide levels,and CT values.CT values may serve as potential imaging markers to assess metabolic risk in SCS patients.
8.Clinical characteristics of clinical and subclinical Cushing's syndrome caused by primary bilateral macronodular adrenal hyperplasia
Huai-Jin XU ; Bing LI ; Kang CHEN ; Hui-Xin ZHOU ; Ya-Jing WANG ; Li ZANG ; Xian-Ling WANG ; Yu CHENG ; Jin DU ; Qing-Hua GUO ; Wei-Jun GU ; Zhao-Hui LYU ; Jian-Ming BA ; Jing-Tao DOU ; Yi-Ming MU
Medical Journal of Chinese People's Liberation Army 2025;50(7):800-807
Objective To investigate the clinical characteristics of patients with clinical and subclinical Cushing's syndrome caused by primary bilateral macronodular adrenal hyperplasia(PBMAH).Methods A retrospective analysis was performed on the clinical data of 198 patients with Cushing's syndrome caused by PBMAH diagnosed in the First Medical Center of Chinese PLA General Hospital from January 2004 to October 2024.According to clinical manifestations,the patients were classified into clinical type Cushing's syndrome(n=61)and subclinical type Cushing's syndrome(n=137),and the clinical characteristics of the two types were compared.Results The mean age at diagnosis of patients with PBMAH-induced Cushing's syndrome was(53.5±10.4)years,including 118 males and 80 females,with a male-to-female ratio of 1.475:1.Compared with the subclinical type,the clinical type had a higher proportion of females,higher levels of serum cortisol,24-hour urine free cortisol(24 h UFC),and inhibited serum cortisol after low-dose dexamethasone suppression.Additionally,the clinical type had lower plasma ACTH,larger adrenal nodules and a higher risk of surgery(P<0.05)compared with those in subclinical type.The incidences of hypertension,dyslipidemia,obesity,diabetes mellitus,hypokalemia,vitamin D deficiency,osteoporosis,coronary heart disease,and cerebrovascular disease in patients with Cushing's syndrome caused by PBMAH were 87.9%,50.5%,37.1%,36.9%,27.8%,25.9%,18.7%,18.7%and 12.1%,respectively.Among them,compared with subclinical type patients,clinical type patients had higher incidence of hypokalaemia,vitamin D deficiency and osteoporosis(P<0.05),while there were no statistically significant differences in the incidences of other comorbidities between the two types(P>0.05).The results of postoperative follow-up for PBMAH patients showed that the short-term biochemical remission rate of unilateral total adrenalectomy was 41.5%(22/53)and the long-term biochemical remission rate was 32.0%(8/25).The short-term biochemical remission rate of unilateral partial(or nodular)adrenalectomy was 52.9%(9/17),and the long-term biochemical remission rate was 14.3%(1/7).All patients who underwent unilateral total adrenalectomy plus contralateral partial resection developed adrenal insufficiency(3/3),and 1 patient(1/3)relapsed 3.4 years after surgery.Conclusion Clinical and subclinical types of Cushing's syndrome caused by PBMAH have their distinct clinical characteristics.Surgery is an effective treatment for PBMAH,but a certain proportion of patients fail to achieve biochemical remission after non-bilateral total adrenalectomy.
9.Effect and mechanism of high-glucose environment on osteoblast function and bone quality in mice
Zhi-Kang GUO ; Xue LI ; Rui WANG ; Xi-Xiu XIE ; Tao-Jin FENG ; Yi LI ; Peng-Bin YIN ; Li-Jun XU ; Li-Xia ZHANG
Medical Journal of Chinese People's Liberation Army 2025;50(10):1306-1314
Objective To explore the effects of different glucose concentrations on the synthesis and secretion of bone collagen in osteoblasts and the impact of diabetes on bone quality in mice.Methods(1)Primary osteoblasts were extracted from the skulls of neonatal mice via collagenase digestion and cultured in four groups under different glucose concentrations:normal glucose(5.5 mmol/L),moderate glucose(11.5 mmol/L),moderate-high glucose(16.5 mmol/L),and high glucose(25 mmol/L).EdU staining was performed to evaluate cell proliferation,while the Transwell assay was used to assess cell migration.Immunofluorescence and Western blotting were performed to detect and quantitatively analyze the content of type Ⅰ collagen(Col-1).Alizarin red S(ARS)staining and alkaline phosphatase(ALP)staining were applied to assess the effects of different glucose concentrations on osteogenic differentiation.(2)Six-week-old male C57BL/6 mice were randomly divided into control group and model group(5 in each group).The model group was fed a high-fat diet for 4 weeks followed by streptozotocin(STZ)injection to establish a diabetic mouse model.The osteogenic differentiation capacity of primary osteoblasts from both groups was assessed.(3)Micro-computed tomography(Micro-CT)was employed to analyze femoral bone mineral density(BMD),bone volume/tissue volume(BV/TV),trabecular number(Tb.N),and trabecular separation(Tb.Sp).Three-point bending test was conducted to evaluate mechanical parameters including maximum load,Young's modulus,fracture energy,and stiffness.RT-qPCR was employed to assess the expression of osteogenic differentiation genes(Alp,Opn,Col1a1,and Lox).Masson staining and Mallory staining were used to evaluate Col-1 content in trabecular bone.Results(1)EdU and Transwell assay results demonstrated that with the gradual increase in glucose concentration,the proliferation and migration abilities of osteoblasts were significantly decreased(P<0.001),and the protein expression levels of Col-1 and lysyl oxidase(LOX)were significantly reduced(P<0.01 or P<0.001).ARS and ALP staining revealed that calcium salt deposition and ALP activity in osteoblasts were significantly decreased with increasing glucose concentration(P<0.05 or P<0.001).(2)Compared with control group,mice in model group exhibited typical"three polies and one weight loss"symptoms(polyuria,polydipsia,polyphagia,and weight loss)of diabetes,and ARS and ALP staining showed a significant reduction in osteoblasts(P<0.001).(3)Micro-CT and three-point bending test results indicated that,compared with control group,mice in model group showed microarchitectural deterioration of bone,decreased Tb.N,increased Tb.Sp,and significantly reduced maximum load,Young's modulus,fracture energy,and stiffness(P<0.05).RT-qPCR results showed that the relative mRNA expression levels of osteogenic differentiation genes(Alp,Opn,Col1a1,and Lox)were significantly decreased in model group compared with control group(P<0.01 or P<0.001).Masson and Mallory staining indicated a significant reduction in collagen content in model group compared with control group(P<0.01).Conclusions High-glucose environment inhibits osteoblast proliferation,differentiation,and migration.Diabetic mice exhibit reduced bone quality and increased bone fragility,potentially mediated by decreased lysyl oxidase and collagen levels.
10.Artesunate attenuating intestinal mucosal barrier injury in acute graft-versus-host disease mice
Jing KANG ; Xu CHENG ; Jin-Miao ZHENG ; Yu-Xue LI ; Li-Zhuang HAN ; Shu YANG
Acta Anatomica Sinica 2025;56(5):566-575
Objective To investigate the mechanism by which artesunate(ART)attenuates intestinal mucosal barrier damage in acute graft-versus-host disease(aGVHD)and the synergistic effect of ART in combination with dexamethasone(DXM)in the treatment of aGVHD mice.Methods The aGVHD mouse model was established by bone marrow haematopoietic stem cell transplantation.The mice were divided into 9 groups,including normal mice control(Ctrl),aGVHD mice(aGVHD),normal mice receiving ART[30 mg/(kg·d)],aGVHD mice receiving low-dose ART[10 mg/(kg·d)],aGVHD mice receiving medium-dose ART[30 mg/(kg·d)],aGVHD mice receiving high-dose ART[50 mg/(kg·d)],aGVHD mice receiving DXM[20 mg/(kg·d)],aGVHD mice receiving ART[30 mg/(kg·d)]and DXM[20 mg/(kg·d)],and aGVHD mice receiving ART[30 mg/(kg·d)]and halved DXM[10 mg/(kg·d)].Survival rate and clinical parameters were assessed.HE staining and Alcian blue-periodic acid-Schiff(AB-PAS)staining were used to observe the histopathological changes in the intestinal mucosa of the mice;Real-time PCR,Western blotting and immunohistochemistry were used to detect the structure of the intestinal mucosal barrier,the T cell differentiation related transcription factors and cytokines,and the key enzymes of energy metabolism.Flow cytometry was used to detect the T helper cell 17(Th17)and regulatory T cells(Treg).Results After 30 days of ART treatment,aGVHD mice showed significant relief of systemic symptoms and increase in survival rate.In aGVHD mice treated with ART,the intestinal mucosal barrier structure was restored,and the intestinal mucosal permeability was reduced.The activity of AMP-activated protein kinase(AMPK)/mTOR pathway was inhibited,and the energy metabolism pattern of T cells was dominated by fatty acid synthesis.The balance of Th17/Treg was restored due to the decrease of Th 17 and the increase of Treg.The effect of ART+DXM treatment on aGVHD mice was comparable to that of DXM treatment alone,and the survival rate of mice was higher.In particular,the recovery of the intestinal mucosal barrier function was most obvious in the mice treated with ART+half-dose DXM.Conclusion ART reduces the immune injury of allo-T cells to the intestinal mucosal barrier by recovering the Th17/Treg balance,thus maintaining the integrity of the intestinal mucosal barrier function.The synergistic effect of ART and DXM combination treatment in aGVHD mice can reduce the incidence of DXM side effects by decreasing the dosage of DXM.


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