ObjectiveTo investigate the effect of Liuwei Dihuangwan drug-containing serum （LDP） on epithelial-mesenchymal transition （EMT） and the expression of cancer stem cell （CSC） properties in 4T1 cells from triple-negative breast cancer by intervening myeloid-derived suppressor cells （MDSCs）. MethodsSPF-grade female SD rats were randomly divided into three groups， which were given 0.39， 1.94， 3.89 g·kg^-1·d^-1 suspension of Liuwei Dihuangwan for 7 days， respectively， to prepare low-， medium-， and high-dose LDPs. 4T1 cells were inoculated subcutaneously into the mammary glands of SPF-grade female Balb/c mice to construct a transplantation tumor model. Bone marrow cells were extracted from the tibia and femur and induced into MDSCs in vitro. The cell counting kit-8 （CCK-8） assay was used to detect the viability of 4T1 cells and MDSCs. The number of MDSCs and the expressions of CSC surface markers CD44 and CD24 in 4T1 cells were detected by flow cytometry （FC）. The migration， invasion， and proliferation of 4T1 cells were detected by cell scratch assay， Transwell invasion assay， and plate colony-forming assay， respectively. Western blot （WB） was used to detect the protein expression of transforming growth factor-β （TGF-β）， nuclear factor-κB （NF-κB）， C-X-C motif chemokine ligand 2 （CXCL2）， E-cadherin， and N-cadherin. The expression of EMT-related proteins E-cadherin and N-cadherin were detected by immunofluorescence （IF）. ResultsCompared with the normal group， LDP showed no significant inhibitory effect on the cell viability of 4T1 cells， but it significantly reduced the viability and number of MDSCs and reduced the number of MDSCs， as well as the expression of TGF-β （P<0.05， P<0.01）. The migration， invasion， and proliferation of 4T1 cells were increased after co-culture with MDSCs （P<0.05， P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin and the proportion of CSC （CD44⁺CD24^-） were elevated （P<0.05， P<0.01）， while the expression of E-cadherin was decreased （P<0.05）. After the intervention of MDSCs with LDP， followed by co-culture with 4T1 cells， the migration， invasion， and proliferation of 4T1 cells were obviously reduced （P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin were decreased （P<0.05， P<0.01）， and the expression of E-cadherin was increased （P<0.05， P<0.01）. There was no statistical difference in the proportion of CSC （CD44⁺CD24^-） in 4T1 cells. However， the proportion of CSC （CD44⁺CD24^-） was decreased in the co-culture system of 4T1 cells and MDSCs with LDP intervention （P<0.05， P<0.01）. ConclusionLDP can reduce the viability and number of MDSCs and the expression of TGF-β， NF-κB， and CXCL2， reverse EMT， and reduce the characteristic expression of CSC to inhibit the migration， invasion， and proliferation of 4T1 cells.

ObjectiveTo investigate the effect of Liuwei Dihuangwan drug-containing serum （LDP） on epithelial-mesenchymal transition （EMT） and the expression of cancer stem cell （CSC） properties in 4T1 cells from triple-negative breast cancer by intervening myeloid-derived suppressor cells （MDSCs）. MethodsSPF-grade female SD rats were randomly divided into three groups， which were given 0.39， 1.94， 3.89 g·kg^-1·d^-1 suspension of Liuwei Dihuangwan for 7 days， respectively， to prepare low-， medium-， and high-dose LDPs. 4T1 cells were inoculated subcutaneously into the mammary glands of SPF-grade female Balb/c mice to construct a transplantation tumor model. Bone marrow cells were extracted from the tibia and femur and induced into MDSCs in vitro. The cell counting kit-8 （CCK-8） assay was used to detect the viability of 4T1 cells and MDSCs. The number of MDSCs and the expressions of CSC surface markers CD44 and CD24 in 4T1 cells were detected by flow cytometry （FC）. The migration， invasion， and proliferation of 4T1 cells were detected by cell scratch assay， Transwell invasion assay， and plate colony-forming assay， respectively. Western blot （WB） was used to detect the protein expression of transforming growth factor-β （TGF-β）， nuclear factor-κB （NF-κB）， C-X-C motif chemokine ligand 2 （CXCL2）， E-cadherin， and N-cadherin. The expression of EMT-related proteins E-cadherin and N-cadherin were detected by immunofluorescence （IF）. ResultsCompared with the normal group， LDP showed no significant inhibitory effect on the cell viability of 4T1 cells， but it significantly reduced the viability and number of MDSCs and reduced the number of MDSCs， as well as the expression of TGF-β （P<0.05， P<0.01）. The migration， invasion， and proliferation of 4T1 cells were increased after co-culture with MDSCs （P<0.05， P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin and the proportion of CSC （CD44⁺CD24^-） were elevated （P<0.05， P<0.01）， while the expression of E-cadherin was decreased （P<0.05）. After the intervention of MDSCs with LDP， followed by co-culture with 4T1 cells， the migration， invasion， and proliferation of 4T1 cells were obviously reduced （P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin were decreased （P<0.05， P<0.01）， and the expression of E-cadherin was increased （P<0.05， P<0.01）. There was no statistical difference in the proportion of CSC （CD44⁺CD24^-） in 4T1 cells. However， the proportion of CSC （CD44⁺CD24^-） was decreased in the co-culture system of 4T1 cells and MDSCs with LDP intervention （P<0.05， P<0.01）. ConclusionLDP can reduce the viability and number of MDSCs and the expression of TGF-β， NF-κB， and CXCL2， reverse EMT， and reduce the characteristic expression of CSC to inhibit the migration， invasion， and proliferation of 4T1 cells.

OBJECTIVE: To standardize the operative procedure for tissue-engineered cartilage repair, by demonstrating surgical technique of arthroscopic implantation of decalcified cortex-cancellous bone scaffolds, and summarizing the surgical experience of the sports medicine department team at Peking University Third Hospital. METHODS: This article elaborates on surgical techniques and skills, focusing on the unabridged implantation technology and surgical procedure of decalcified cortex-cancellous bone scaffolds under arthroscopy: First, the patient was placed in the supine position. After anesthesia had been established, the surgeon established an arthroscope and explored the damaged area under the scope. After confirming the size and location of the injury site, the surgeon cleaned the damaged cartilage, and also trimmed the edges of the cartilage to ensure that the cut surface was smooth and stable. the surgeon performed the micro-fracture surgery in the area of cartilage injury, and then measured the size of the injured area under the scope. Next, the surgeon manually trimmed the tissue-engineered scaffold based on the measurements taken under the arthroscope, and then directly implanted the scaffold using a sleeve. A honeycomb-shaped fixator was used to implant absorbable nails to fix the scaffold. After the scaffold was installed, the knee was repeatedly flexed and extended for 10-20 times to ensure stability and range of motion. Finally, the arthroscope was withdrawn and the wound was closed. RESULTS: Decalcified cortex-cancellous bone scaffolds possessed unparalleled advantages over synthetic materials in terms of morphology and biomechanics. The cancellous bone part of the scaffold provided a three-dimensional, porous space for cell growth, while the cortical bone part offered the necessary mechanical strength. The surgery was performed entirely under arthroscopy to minimize invasiveness to the patient. Absorbable pins were used for fixation to ensure the stability of the scaffold. This technique could effectively improve the prognosis of the patients with cartilage injuries and standardized the surgical procedures for arthroscopic tissue-engineered scaffold operations in the patients with cartilage damage. CONCLUSION With the standard arthroscopic tissue-engineered scaffold repair technique, it is possible to successfully repair damaged cartilage, alleviate symptoms in the short term, and provide a more ideal long-term prognosis. The author and their team explain the surgical procedures for tissue-engineered scaffolds under arthroscopy, with the aim of guiding future clinical practice.
Tissue Engineering/methods* ; Humans ; Tissue Scaffolds ; Arthroscopy/methods* ; Cartilage, Articular/surgery*

OBJECTIVE: To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). METHODS: The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. RESULTS: In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). CONCLUSION Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.
Abietanes/therapeutic use* ; Osteosarcoma/enzymology* ; Cisplatin/therapeutic use* ; Humans ; Cell Line, Tumor ; Animals ; Apoptosis/drug effects* ; Mice, Nude ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MAP Kinase Signaling System/drug effects* ; Bone Neoplasms/enzymology* ; Cell Cycle/drug effects* ; Xenograft Model Antitumor Assays ; Mice ; Drug Resistance, Neoplasm/drug effects* ; Neoplasm Invasiveness ; Mice, Inbred BALB C

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

The differences in chemical quality characteristics between Xinjiang Rehmannia glutinosa and R. glutinosa were analyzed to provide a theoretical basis for the introduction and quality control of R. glutinosa. In this study, the high performance liquid chromatography(HPLC) fingerprints of 6 batches of Xinjiang R. glutinosa and 10 batches of R. glutinosa samples were established. The content of iridoid glycosides, phenylethanoid glycosides, monosaccharides, oligosaccharides, and polysaccharides in Xinjiang R. glutinosa and R. glutinosa was determined by high performance liquid chromatography-diode array detection(HPLC-DAD), high performance liquid chromatography-evaporative light scattering detection(HPLC-ELSD), and ultraviolet-visible spectroscopy(UV-Vis). The determination results were analyzed with by chemical pattern recognition and entropy weight TOPSIS method. The results showed that there were 19 common peaks in the HPLC fingerprints of the 16 batches of R. glutinosa, and catalpol, aucubin, rehmannioside D, rehmannioside A, hydroxytyrosol, leonuride, salidroside, cistanoside A, and verbascoside were identified. Hierarchical cluster analysis(HCA) and principal component analysis(PCA) showed that Qinyang R. glutinosa, Mengzhou R. glutinosa, and Xinjiang R. glutinosa were grouped into three different categories, and eight common components causing the chemical quality difference between Xinjiang R. glutinosa and R. glutinosa in Mengzhou and Qinyang of Henan province were screened out by orthogonal partial least squares discriminant analysis(OPLS-DA). The results of content determination showed that there were glucose, sucrose, raffinose, stachyose, polysaccharides, and nine glycosides in Xinjiang R. glutinosa and R. glutinosa samples, and the content of catalpol, rehmannioside A, leonuride, cistanoside A, verbascoside, sucrose, and glucose was significantly different between Xinjiang R. glutinosa and R. glutinosa. The analysis with entropy weight TOPSIS method showed that the comprehensive quality of R. glutinosa in Mengzhou and Qinyang of Henan province was better than that of Xinjiang R. glutinosa. In conclusion, the types of main chemical components of R. glutinosa and Xinjiang R. glutinosa were the same, but their content was different. The chemical quality of R. glutinosa was better than Xinjiang R. glutinosa, and other components in R. glutinosa from two producing areas and their effects need further study.
Rehmannia/classification* ; Drugs, Chinese Herbal/chemistry* ; Chromatography, High Pressure Liquid/methods* ; Quality Control

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Objective To investigate the mechanism of Kuijie Ankang Decoction in regulating immune,autophagy and intestinal flora in the treatment of ulcerative colitis(UC).Methods UC mouse model was established by free drinking with sodium dextran sulfate.The mice were randomly divided into blank control group,model group,Kuijie Ankang Decoction group,salazine sulfopyridine(SASP)group and 3-methyladenine(3-MA)group,with 12 mice in each group.Each drug group was given corresponding drugs for gavage,the blank control group and model group were given the same volume of distilled water for gavage for 7 days.The general condition of mice was observed and the disease activity index(DAI)was scored,the morphology of colon tissue was observed by HE staining,the contents of TNF-α,IL-1β,IL-6,IL-8 and IL-10 in colon tissue were detected by ELISA,the mRNA and protein expressions of LC3,Beclin-1 and p62 in colon tissue were detected by RT-qPCR and Western blot,respectively.16S rDNA sequencing was used to analyze the structure of intestinal flora.Results Compared with the blank control group,the mice in the model group showed a decrease in body mass,an increase in DAI score,a decrease in colon length,serious mucosal injury and inflammatory cell infiltration,the contents of TNF-α,IL-1β,IL-6 and IL-8 in colon tissue significantly increased(P<0.05,P<0.01),the content of IL-10 decreased(P<0.01),the mRNA expressions of LC3 and Beclin-1 in colon tissue decreased(P<0.05,P<0.01),the mRNA and protein expression of p62 increased(P<0.01),while the expressions of LC3Ⅱ/LC3Ⅰ and Beclin-1 proteins decreased(P<0.05,P<0.01).Compared with the model group,the body mass of mice in Kuijie Ankang Decoction group and SASP group increased(P<0.05),DAI score decreased(P<0.05),the colon length increased(P<0.05),the pathological damage of colon mucosa was alleviated,the contents of TNF-α,IL-1β,IL-6 and IL-8 in colon tissue decreased(P<0.05,P<0.01),the content of IL-10 increased(P<0.01),the expressions of LC3 and Beclin-1 mRNA in colon tissue increased(P<0.05,P<0.01),the expression of p62 mRNA and protein decreased(P<0.05,P<0.01),the expressions of LC3Ⅱ/LC3Ⅰand Beclin-1 protein increased(P<0.05,P<0.01).16S rDNA sequencing results showed that the diversity and evenness of the intestinal flora in the model group mice decreased,with a decrease in the relative abundance of Firmicutes,Actinobacteria and Patescibacteria(P<0.05),and an increase in the relative abundance of Bacteroidota,Verrucomicrobiota and Proteobacteria(P<0.05);the relative abundance of Bacilli and Coriobacteriia decreased(P<0.05),the relative abundance of Bacteroidia,Clostridia and Verrucomicrobiae increased(P<0.05);the relative abundance of Ligilactobacillus and Dubosiella decreased(P<0.05),the relative abundance of unclassified Muribaculaceae,Lachnospiraceae NK4A136_group,Akkermansia and unclassified Lachnospiraceae increased(P<0.05).Compared with the model group,the diversity and evenness of intestinal flora increased in Kuijie Ankang Decoction group and SASP group,with an increase in the relative abundance of Firmicutes(P<0.05),a decrease in the relative abundance of Bacteroidota and Verrucomicrobiota(P<0.05),the relative abundance of Bacteroidia and Bacilli increased(P<0.05),the relative abundance of Verrucomicrobiae decreased(P<0.05);the relative abundance of unclassified Muribaculaceae and Ligilactobacillus increased(P<0.05),while the relative abundance of Lachnospiraceae NK4A136_group and Akkermansia decreased(P<0.05).Conclusion Kuijie Ankang Decoction can significantly improve the intestinal mucosal injury of UC mice,and the mechanism may be related to the regulation of colon autophagy level and intestinal flora disorder.