1.Tumor-associated macrophages affect breast cancer endocrine resistance through FOXM1/Wnt/ β-catenin pathway
Qilong ZHAO ; Zejie SHAO ; Qingshan MA ; Xin WANG ; Changxin LYU
Chinese Journal of Endocrine Surgery 2023;17(4):484-489
Objective:To explore the influence of tumor-associated macrophages (TAMs) on endocrine resistance in breast cancer through the forkhead box M1 (FOXM1) /Wnt/ β-catenin pathway. Methods:Tamoxifen-resistant breast cancer cells were cultured, THP-1 cells were induced into macrophages (MΦ), and further induced into TAMs. After being cultured in the conditioned medium (CM) of MCF-7 cells for 24 hours, MΦ were defined as MS cells. After being cultured in the CM of MCF-7R cells for 24 hours, MΦ were defined as MR cells. MCF-7 cells, after being cultured in the CM of macrophages for 24 hours, were defined as MCF-7 (MΦ) cells. MCF-7 cells, after being cultured in the CM of MS cells for 24 hours, were defined as MCF-7 (MS) cells. MCF-7 cells, after being cultured in the CM of MR cells for 24 hours, were defined as MCF-7 (MR) cells. Cell viability and invasion ability were evaluated using CCK-8 and Transwell assays. The protein levels of CD163, Wnt1, β-catenin, and FOXM1 in different groups were examined by qRT-PCR and Western blot. Results:Compared to the MS group (mRNA: 1.49±0.12, protein: 1.15±0.12), CD163 expression was higher in the MR group (mRNA: 2.33±0.16, protein 1.52±0.11) ( t=7.28, P=0.002) ( t=3.94, P=0.017), indicating that tamoxifen-resistant breast cancer cells can induce polarization of more MΦ into TAMs. TAMs increased the expression of FOXM1 in breast cancer cells, which further activated the Wnt/ β-catenin pathway. Compared to the MCF-7 (MΦ) group, the MCF-7 (MS) and MCF-7 (MR) groups showed enhanced cell viability and invasion, with the most significant increase observed in the MCF-7 (MR) group. Compared with MCF-7 (MΦ) cells, the levels of Wnt1, β-catenin, and FOXM1 in MCF-7 (MS) and MCF-7 (MR) cells were significantly increased, with the highest levels observed in the MCF-7 (MR) group with the most TAM polarization. Compared to the MCF-7 group, both the MCF-7 (MR) and MCF-7+pcDNA-FOXM1 groups showed increased levels of Wnt1 and β-catenin, enhanced cell viability and invasion. Compared to the MCF-7 (MR) group, the MCF-7 (MR) + si-FOXM1 group showed reduced levels of Wnt1 and β-catenin, weakened cell viability and invasion. Conclusion:TAMs promote endocrine resistance in breast cancer by upregulating FOXM1 and activating the Wnt/ β-catenin pathway.
2.Network pharmacology and molecular docking to study the mechanism of action of alpinumisoflavone in a temporomandibular joint osteoarthritis cell model
Journal of Prevention and Treatment for Stomatological Diseases 2024;32(8):578-588
Objective:
To explore the potential role of alpinumisoflavone (AIF) in the treatment of temporomandibular joint osteoarthritis (TMJOA) cell model through network pharmacology and molecular docking and to provide a research basis for AIF in the treatment of TMJOA.
Methods:
GeneCards, OMIM, DisGeNET, and PharmGKB databases were used to screen TMJOA disease targets, and PharmMapper and HERB were used to retrieve AIF-related targets. The intersection targets of the compounds and diseases were uploaded to the STRING database to obtain the key targets for GO and KEGG enrichment analysis, while the key targets in related signaling pathways were evaluated through molecular docking. Approval was obtained from the Ethics Committee to extract condylar chondrocytes from 3-week-old SD rats. The CCK-8 assay was used to detect AIF cytotoxicity on condylar chondrocytes. Condylar chondrocytes were induced with 10 ng/mL interleukin 1β (IL-1β) for 24 h to construct a TMJOA cell model. The experiment was divided into three groups: control group, comprising condylar chondrocytes cultured in DMEM for 48 h; IL-1β group, comprising condylar chondrocytes pre-cultured in DMEM for 24 h, after which IL-1β was added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h; and the IL-1β+10 μmol/L AIF group, comprising condylar chondrocytes pre-cultured in DMEM medium containing 10 μmol/L AIF for 24 h, after which IL-1β was added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h. The effect of AIF on condylar chondrocyte apoptosis in the TMJOA cell model was detected by flow cytometry. The experiment was divided into four groups: control group, IL-1β group, IL-1β+10 μmol/L AIF group, and IL-1β+30 μmol/L AIF group. The IL-1β+30 μmol/L AIF group was pre-cultured in DMEM containing 30 μmol/L AIF for 24 h, after which IL-1β was added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h. The remaining three groups were cultured in the same manner as before. The mRNA and protein expression of apoptosis-associated B-cell leukemia/lymphoma-2 (Bcl2), cysteinyl aspartate specific protease 3 (caspase-3), matrix degradation-associated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 13 (MMP13) were detected by qPCR and western blot, by AIF in the TMJOA cell model.
Results:
The PharmMapper and HERB database search yielded 300 AIF compound targets. The GeneCards, OMIM, DisGeNET, and PharmGKB databases yielded 378 TMJOA disease targets. Thirty-three potential common targets were obtained by intersecting compounds with disease targets. The common targets were uploaded into the STRING database to obtain 31 key targets that were mainly associated with apoptosis and extracellular matrix degradation. This process may be associated with the MAPK, estrogen, and TNF signaling pathways. The molecular docking results showed that AIF has good binding activity with extracellular signal-regulated kinase 1/2 (ERK1/2) and estrogen receptor gene 1/2 (ESR1/2), which are key targets in the MAPK and estrogen signaling pathways. The CCK-8 assay showed that AIF had no obvious cytotoxicity to condylar chondrocytes. The cell experiments showed that AIF inhibited apoptosis in the IL-1β+10 μmol/L AIF group compared to the IL-1β group. Compared to the IL-1β group in the IL-1β+10 μmol/L AIF group and the IL-1β+30 μmol/L AIF group, AIF upregulated Bcl2 and downregulated caspase-3 mRNA and protein expression and inhibited ADAMTS4, MMP3, and MMP13 mRNA and protein expression.
Conclusion
AIF inhibited apoptosis in the TMJOA cell model by upregulating Bcl2 and downregulating caspase-3 mRNA and protein expression, and inhibited extracellular matrix degradation induced by IL-1β, thereby delaying TMJOA progression.
3.Constructing an animal model of temporomandibular joint osteoarthritis in Sprague-Dawley rats by digital technology
Penghui LIU ; Fan WU ; Zejie WANG ; Gaoyi WU ; Libo ZHOU
Chinese Journal of Tissue Engineering Research 2024;28(35):5642-5648
BACKGROUND:Temporomandibular joint osteoarthritis is a common oral disease with a high incidence.However,temporomandibular joint osteoarthritis is not easy to be detected in the early stage,and it is difficult to obtain clinical pathological specimens,so it is difficult to carry out related research.The application of digital 3D printing technology to animal models of Temporomandibular joint osteoarthritis increases the consistency of the animal models,thus promoting the study of temporomandibular joint osteoarthritis. OBJECTIVE:To establish a standardized animal model of temporomandibular joint osteoarthritis using novel digital technology. METHODS:According to the different modeling methods of unilateral anterior crossbite,30 female Sprague-Dawley rats were randomly divided into traditional model group,digital model group,and control group(n=10 per group).Cartilage specimens of the condyles were collected at 4 and 8 weeks after modeling.The apparent morphology was observed by stereoscopic microscope.The pathological morphology was observed by hematoxylin-eosin staining and Safranin O/fast green staining.Changes in the expression of interleukin-1β and tumor necrosis factor-α were observed by ELISA,and changes in the expression of aggrecan,type Ⅱ collagen and matrix metalloproteinase-13 were observed by immunohistochemical staining. RESULTS AND CONCLUSION:Different degrees of degeneration were observed in the digital and traditional model groups.The body mass of rats in both the model groups decreased during the 1st week after intervention and subsequently demonstrated growth trend and were significantly lower than that in the control group.The results of stereoscopic microscope showed that at 4 and 8 weeks after modeling,the deformation and defect degree of the digital model group was significantly higher than that of the traditional model group.At these two time points,the Osteoarthritis Research Society International scores of the digital model group and the traditional model group were higher than those of the control group,and the Osteoarthritis Research Society International score of the digital model group was higher than that of the traditional model group(P<0.05).Histopathological observation showed that the modified Mankin score and Osteoarthritis Research Society International score of the two model groups were significantly higher than those of the control group of the same age at 4 and 8 weeks after modeling(P<0.05).Immunohistochemical staining results showed that at two time points,compared with the control group of the same age,the expression of aggrecan and type Ⅱ collagen decreased in the traditional model group and the digital model group,while the expression of matrix metalloproteinase 13 increased(P<0.05).ELISA results showed that the expression levels of inflammatory factors interleukin-1β and tumor necrosis factor-α in the traditional and digital model groups were higher than those in the control group at 8 weeks,and the expression levels of interleukin-1β and tumor necrosis factor-α in the digital model group were higher than those in the traditional model group(P<0.05).To conclude,the personalized metal tube designed and produced by 3D printing technology can quickly guide the osteoarthritis-like lesions of the temporomandibular joint without repeated trial and adjustment,which is reproducible and suitable for promotion and application.
4.Effects of bisdemethoxycurcumin promoting neuronal differentiation of neuroblastoma cells in mice and its mechanism
Jiaxin WANG ; Hongzhi FANG ; Min WU ; Zejie YANG ; Wenbo XU ; Shuang ZHANG ; Shali LI ; Genyun TANG
China Pharmacy 2024;35(5):578-583
OBJECTIVE To study the effects of the curcumin derivative bisdemethoxycurcumin (BC) promoting neuronal differentiation of neuroblastoma cells Neuro-2a (N2a) in mice and its mechanism. METHODS The effects of BC (1, 2, 4, 6, 8, 10 μmol/L) on the viability of N2a cells were detected by MTT assay to determine the concentration range of drug treatment. The control group, retinoic acid (RA) group (10 μmol/L) and BC groups (1, 2 and 4 μmol/L) were set up, and the length of neural protrusions of the differentiated cells was measured and the cell differentiation rate was calculated after 48 h and 72 h of culture. Compared with 0 min group, Western blot was used to detect the phosphorylation levels of protein kinase B (Akt), extracellular- signal regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38) proteins in cells treated by 4 μmol/L BC for 5, 15, 30, 60, 120 min. After intervention with inhibitors LY294002 (LY) and PD98059 (PD), the effects of BC on Akt and ERK1/2 protein phosphorylation levels and promoting neural differentiation were further validated. RESULTS According to the MTT experiment, the BC concentrations for subsequent induction of cell differentiation were determined to be 1, 2, and 4 μmol/L. After 48 hours of differentiation, compared with the control group, the cell differentiation rate in RA group and BC 1, 2 and 4 μmol/L groups, the length of cellular neural processes wjxhhxx413@163.com in the BC 4 μmol/L group significantly increased (P<0.05 or P<0.01);after inducing differentiation of BC for 72 hours,compared with the control group, the cell differentiation rate and the length of cellular neural processes in the RA group, the cell differentiation rate in the BC 4 μmol/L group, and the length of cellular neural processes in the BC 2 μmol/L group all significantly increased (P<0.05 or P<0.01).Compared with the 0 min group, the phosphorylation levels of Akt, ERK1/2, and p38 proteins in cells of the 5, 15, 30, 60 and 120 min groups increased to varying degrees after treated by 4 μmol/L BC, and some differences were statistically significant (P<0.05 or P<0.01). After adding the inhibitor LY/PD, compared with the BC group, the phosphorylation level of ERK1/2 protein in the PD+BC group cells were significantly reduced (P<0.01), and the cell differentiation rates in the LY group, LY+BC group, PD group, and PD+BC group was significantly reduced (P<0.01). CONCLUSIONS BC promotes N2a cell differentiation mainly by increasing cell differentiation rate and neural protrusion length. The mechanism may be related to the activation of mitogen-activated protein kinase/ ERK and PI3K/Akt signaling pathways.
5. Novel mechanisms driving renal tubulointerstitial fibrosis
Yanni ZHANG ; Yuxin DUAN ; Yi BAI ; Jinyao YU ; Jiayi SUN ; Zejie WANG ; Ling LI ; Qifa YE
Chinese Journal of Clinical Pharmacology and Therapeutics 2024;29(3):348-353
Renal fibrosis, especially tubulointerstitial fibrosis, is the most common pathway of all chronic kidney diseases progressing to end-stage renal diseases. Several adaptive reactions occur in renal tubular epithelial cells after chronic injury, such as changes in glycolipid metabolism, unfolded protein response, autophagy and senescence, epithelial-to-mesenchymal transition and G2/M cell cycle arrest. Maladaptive repair mechanisms can induce tubulointerstitial fibrosis. This article will discuss the molecular mechanism of these adaptive responses of renal tubular epithelial cells driving renal tubulointerstitial fibrosis, and provide a basis for exploring new drug targets for renal tubulointerstitial fibrosis.