scholarly journals Hellebrigenin anti-pancreatic cancer effects based on apoptosis and autophage

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9011
Author(s):  
Xiaolu Wei ◽  
Jing He ◽  
Bo Gao ◽  
Lingyu Han ◽  
Yingqiu Mao ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Morphological Observation ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Toad Skin ◽  
Significant Difference ◽  
Early Apoptosis

Hellebrigenin is a natural product found in the toad skin secretions and plants of Urginea, including Hellebores and Kalanchoe genera. It has been shown to be active against Leishmania chagasi promastigotes and Trypanosoma cruzi trypomastigotes and also reported to play an anti-tumor effect on several cancer cell lines in vitro, including pancreatic cancer. This study is aimed to investigate the effects of Hellebrigenin on pancreatic carcinoma cells, SW1990 and BxPC-3 in vitro and its molecular mechanism involved in antitumor activities. Our results showed that Hellebrigenin effectively inhibited the proliferation of SW1990 and BxPC-3 cells in dose- and time-dependent manner. Flow cytometry results showed that Hellebrigenin induced the G0/G1 arrest in both of SW1990 and BxPC-3 cells and promoted cell early apoptosis and autophagy according to morphological observation. Immunofluorescence staining results further confirmed that cell apoptosis and autophagy also increased upon the Hellebrigenin treatment. Moreover, higher dose of Hellebrigenin further increased the cell apoptosis rate while decrease the mitochondrial membrane potential 24 h after treatment. The autophagy rate increased 48 h after treatment with significant difference (P < 0.05). Western blot analysis showed that the expression of caspase 3, 7, cleaved caspase 7, Atg 12, LC3 proteins were increased in SW1990 cell after treatment with Hellebrigenin. In addition, increasing expression of caspase 3, 7, 9, PARP, cleaved caspase 3, 7, 9, PARP, the sub basic protein of the PI3K family, Beclin-1, LC 3, Atg 3, 5, 12, 16 L were also observed after BxPC-3 cells treated with Hellebrigenin. In summary, this study reported for the first time that Hellebrigenin effectively induced autophagy and apoptosis especially the early apoptosis in SW1990 and BxPC-3 cells.

scholarly journals Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

2019 ◽  
Vol 2 (22.2) ◽  
pp. 155-164
Author(s):  
Liang Zhang
Keyword(s):  
Methylene Blue ◽  
Cell Apoptosis ◽  
Annulus Fibrosus ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood. Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro. Study Design: This study used an experimental design. Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University. Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 μg/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor β1 [TGF-β1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting. Results: Our results indicated that MB reduced cell viability in a concentration- and timedependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells. Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols. Key words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine

The Study of Effect and Mechanism by Vitamin K2 on Human MDS Cell Line MUTZ-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4357-4357
Author(s):  
Bao-An Chen ◽  
Xin Xu ◽  
Ze-Ye Shao ◽  
Jia-Hua Ding ◽  
Guo-Hua Xia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Caspase 3 ◽  
Cellular Proliferation ◽  
Vitamin K2 ◽  
Time Dependent ◽  
Control Group ◽  
Dependent Manner ◽  
Significant Difference

Abstract The myelodysplastic syndromes (MDS) are characterized by hemopoietic insufficiency associated with cytopenias leading to serious morbidity and the additional risk of leukemic transformation. Vitamin K2(VK2) is reported to induce apoptosis or differentiation of leukemic cell lines in vitro. For investigating the effects and mechanism of VK2 on human MDS cell line MUTZ-1 in vitro,we observed the changes of morphologic features of MUTZ-1 cells by exposing to VK2.The transmission electron microscope was used to observe the apoptosis of MUTZ-1 cells. Cellular proliferation was determined by the MTT assay. The flow cytometry was used to analysis apoptosis rate and the change of cell cycle. The expression of apoposis-related genes bcl-2, survivin and bax were detected by reverse transcriptase polymerase chain reaction(RT-PCR).The activity of caspase-3 was detected by chemiluminescence assay. After exposing to 10μmol L−1 and higher concentration of VK2, it could inhibit MUTZ-1 cells proliferation in a dose-and time-dependent manner(p&lt;0.05). At concentration of 5μmol/l VK2 treatment, it might accelerate cellular proliferation, but there’s no significant difference compared with control group. Apoptosis peak on FCM and positive Annexin-V FITC/PI on cell membrane showed that VK2 induced apoptosis of MUTZ-1 cells in a dose-and-time-dependent manner, G0/G1 cell cycle arrest, significantly dow-regulated the expression of bcl-2 and survivin, but had no effect on the expression of bax.The activities of caspase-3 were significantly increased. Low concentration of VK2 could facilitate cell proliferation. The higher concentration of VK2 could induce apoptosis of MUTZ-1 cells. These results indicate that VK2 induces MUTZ-1 cells apoptosis by activating caspase-3 pathway, the apoptosis related genes bcl-2, survivin down-regulated might play an important role in this process.

Immunomodulatory activities of whey fractions in efferent prefemoral lymph of sheep

1996 ◽  
Vol 63 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Chun W. Wong ◽  
Geoffrey O. Regester ◽  
Geoffrey L. Francis ◽  
Dennis L. Watson
Keyword(s):  
Immune Responses ◽  
Bovine Milk ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Milk Whey ◽  
Significant Difference ◽  
Lymphocyte Phenotypes ◽  
Dose Dependent

SummaryStudies on the immunomodulatory activities of ruminant milk and colostral whey fractions were undertaken. By comparing with boiled colostral whey in a preliminary experiment, a putative heat-labile immunostimulatory factor for antibody responses was found to be present in ovine colostral whey. Studies were then undertaken in sheep in which the efferent prefemoral lymphatic ducts were cannulated bilaterally, and immune responses in the node were measured following subcutaneous injection in the flank fold of whey protein preparations of various purities. A significant sustained decline of efferent lymphocyte output was observed following injection with autologous crude milk whey or colostral whey preparations, but no changes were observed in interferon-gamma levels in lymph plasma. Two bovine milk whey fractions (lactoperoxidase and lactoferrin) of high purity were compared in bilaterally cannulated sheep. A transient decline over the first 6 h was seen in the efferent lymphocyte output and lymph flow rate after injection of both fractions. A significant difference was seen between the two fractions in interferongamma levels in lymph at 6 h after injection. However, no significant changes in the proportion of the various efferent lymphocyte phenotypes were seen following either treatment. Whereas both fractions showed a significant inhibitory effect in a dose-dependent manner on the proliferative response of T lymphocytes, but not B lymphocytes, to mitogenic stimulation in vitro, no similar changes were seen following in vivo stimulation with these two fractions.

Hyperglycemia Promotes Pancreatic Cancer Initiation and Progression by Activating the Wnt/β-Catenin Signaling Pathway

2021 ◽  
Vol 21 ◽  
Author(s):  
Huiming Chen ◽  
Junfeng Zhao ◽  
Ningning Jiang ◽  
Zheng Wang ◽  
Chang Liu
Keyword(s):  
Pancreatic Cancer ◽  
Signaling Pathway ◽  
Precancerous Lesions ◽  
Clinical Specimen ◽  
Ductal Adenocarcinoma ◽  
Dependent Manner ◽  
Pancreatic Cancer Cells ◽  
Cancer Initiation

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, with a 5-year survival rate of less than 10% because of the limited knowledge of tumor-promoting factors and their underlying mechanism. Diabetes mellitus (DM) and hyperglycemia are risk factors for many cancers, including PDAC, that modulate multiple downstream signaling pathways, such as the wingless/integrated (Wnt)/β-catenin signaling pathway. However, whether hyperglycemia promotes PDAC initiation and progression by activating the Wnt/β-catenin signaling pathway remains unclear. Methods: In this study, we used bioinformatics analysis and clinical specimen analysis to evaluate the activation states of the Wnt/βcatenin signaling pathway. In addition, colony formation assays, Transwell assays and wound-healing assays were used to evaluate the malignant biological behaviors of pancreatic cancer cells (PCs) under hyperglycemic conditions. To describe the effects of hyperglycemia and the Wnt/β-catenin signaling pathway on the initiation of PDAC, we used pancreatitis-driven pancreatic cancer initiation models in vivo and pancreatic acinar cell 3-dimensional culture in vitro. Results: Wnt/β-catenin signaling pathway-related molecules were overexpressed in PDAC tissues/cells and correlated with poor prognosis in PDAC patients. In addition, hyperglycemia exacerbated the abnormal activation of β-catenin in PDAC and enhanced the malignant biological behaviors of PCs in a Wnt/β-catenin signaling pathway-dependent manner. Indeed, hyperglycemia accelerated the formation of pancreatic precancerous lesions by activating the Wnt/β-catenin signaling pathway in vivo and in vitro. Conclusion: Hyperglycemia promotes pancreatic cancer initiation and progression by activating the Wnt/β-catenin signaling pathway.

P–061 Protective effect of melatonin against bleomycin, etoposide, and cisplatin (BEP) chemotherapy-induced testicular toxicity in Wistar rats: A biochemical, immunohistochemical and apoptotic genes based evidence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Moradi ◽  
A Faramarzi ◽  
N Goodarzi ◽  
A H Hashemian ◽  
H Cheraghi ◽  
...  
Keyword(s):  
Testosterone Level ◽  
Wistar Rats ◽  
Caspase 3 ◽  
Treated Group ◽  
Serum Testosterone Level ◽  
Dependent Manner ◽  
Qrt Pcr ◽  
Tnf Α ◽  
Significant Difference ◽  
Bep Chemotherapy

Abstract Study question Does exogenous melatonin (MLT) attenuate BEP-induced damage in testicular cells and spermatogenesis in a dose-dependent manner? Summary answer Melatonin protected the testes against BEP-induced testis damage through ameliorating nitro-oxidative stress, apoptosis, and inflammation. However, there was no significant difference between melatonin-treated groups. What is known already Recently, the prevalence of testicular cancer (TC), accounting for the most common cancer among young people of reproductive age (15–40 years), has risen internationally. BEP chemotherapy has increased the 5-year survival rate of TC patients at all stages of testicular germ cell tumors to 90–95%. However, BEP creates a high incidence of male infertility and even long-term genotoxic effects, which emerges as a critical health issue. Melatonin is a well-known potent antioxidant with widespread clinical applications that recently has been giving increasing attention to its role in male sub/infertility. Study design, size, duration 60 Adult male Wistar rats were randomly assigned to six groups (n = 10/group). Group 1, 3, and 4 were injected with vehicle, 10 and 20 mg/kg of melatonin, respectively. Other groups received one cycle of bleomycin, etoposide, and cisplatin for a total of 3 weeks with or without melatonin. Melatonin administration started daily one week before BEP initiation continued on days 2, 9, and 16; and one week after the completion of the BEP cycle. Participants/materials, setting, methods Bodyweight, testes weight, Sperm parameters (count, motility, viability, and morphology), testosterone hormone level, testicular histopathology, stereological parameters, testicular level of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC), the expression of Bcl–2, Bax, Caspase–3, p53, and TNF-α (Real-time PCR and immunohistochemistry) were evaluated at the end of the study (day 35). Main results and the role of chance Our findings showed that melatonin restores the BEP-induced reduction in the body and testes weight (P&lt;.05). the evaluation of quantitative analysis of the testes stereological procedures, QRT-PCR examination and immunohistochemical (IHC) staining revealed that melatonin reverses the BEP-induced impaired spermatogenesis (P&lt;.05). Furthermore, melatonin rectifies BEP-induced disturbance on sperm count, motility, viability, and morphology. The testosterone level in the BEP-treated group was decreased significantly by comparison with the control group (P&lt;.01). By contrast, co-administration of 10 and 20 mg/kg of melatonin could enhance the serum testosterone level significantly (P&lt;.05). Moreover, melatonin enhanced the antioxidant status of the testis by elevating TAC and ameliorating MDA and NO levels. More notably, QRT-PCR examination indicated that melatonin therapy suppressed BEP-induced apoptosis by modulating apoptosis-associated genes such as Bcl–2, Bax, Caspase–3, p53 in the testis (P&lt;.01). Besides, Co-administration of 10 and 20 mg/kg of melatonin with BEP regimen decreased significantly the population of p53 (54.21 ±6.18% and 51.83±8.45, respectively) and TNF-α positive cells (42.91±9.92% and 33.57±2.97, respectively) by comparison to the BEP group. Also, melatonin with low and high doses could enhance the expression of Bcl–2 protein in spermatogenic cells line (59.19±10.18%, 63.08±5.23, respectively) compared to the BEP-treated group. Limitations, reasons for caution Owing to limited laboratory facilities we were not able to perform further studies to verify the mechanism of melatonin in the specific targets by using transfection technique and transgenic. Wider implications of the findings: These findings can draw attention to the clinical application of melatonin and also suggest that melatonin may be an attractive agent for attenuating chemotherapy-associated male sub/infertility. This indolamine also may shorten the fertility recovery period in patients undergoing chemotherapy with the BEP regimen. Trial registration number N/A

Synergistic Apoptotic Effect of Naringenin on enhancing the Anti-Glioma Efficacy of Temozolomide in an in vitro Experimental Model

2021 ◽  
Vol 16 (10) ◽  
pp. 43-49
Author(s):  
Precilla S. Daisy ◽  
S. Kuduvalli Shreyas ◽  
R. Sathish ◽  
T.S. Anitha
Keyword(s):  
Drug Treatment ◽  
Caspase 3 ◽  
Treatment Strategies ◽  
Glioma Cells ◽  
C6 Glioma ◽  
Mitigation Strategies ◽  
Treatment Modalities ◽  
C6 Glioma Cells ◽  
Dependent Manner

Glioma is one of the most devastating and difficult-totreat brain tumors with a very poor prognosis. Despite the current treatment modalities, the overall survival rate is only 5% contributing to a high mortality rate. Nevertheless, of emerging treatment strategies, there is still a rising need for novel mitigation strategies to counteract glioma aggressiveness. One attempt towards this long-term goal was made in this study to reveal the combined efficacy of naringenin, a bioactive flavonoid on enhancing the anti-glioma potency of temozolomide in C6 glioma cells. The cytotoxic effect of temozolomide and naringenin, both individually and in combination was assessed by employing MTT assay. The synergistic effect of the drugs temozolomide and naringenin was determined by calculating the combination index. To confirm the presence of apoptotic changes in the cells at morphological level, acridine orange/ethidium bromide staining was performed. Further, the modulatory effects of the drugs on apoptotic genes, caspase-3 and BCL-2 were evaluated using quantitative real time-PCR. Interestingly, we found that the combinatorial drug treatment was in consensus and effectively inhibited the growth of C6 glioma cells in a dose-dependent manner. Furthermore, this combinatorial drug treatment significantly up-regulated the expression of the proapoptotic gene, caspase-3 and down-regulated the anti-apoptotic gene BCL-2 suggesting a shift of equilibrium towards apoptosis. Our findings suggest that naringenin can be employed as a potent drug to enhance the anti-glioma efficacy of temozolomide and could be therapeutically exploited for the management of glioma.

scholarly journals Metformin Inhibited Growth, Invasion and Metastasis of Esophageal Squamous Cell Carcinoma in Vitro and in Vivo

2018 ◽  
Vol 51 (3) ◽  
pp. 1276-1286 ◽  
Author(s):  
Feng Liang ◽  
Yu-Gang Wang ◽  
Changcheng Wang
Keyword(s):  
Squamous Cell Carcinoma ◽  
Plasma Glucose Level ◽  
Caspase 3 ◽  
Time Dependent ◽  
Dependent Manner ◽  
Metformin Treatment ◽  
Invasion And Migration ◽  
And Migration

Background/Aims: This study aimed at investigating the effects of metformin on the growth and metastasis of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo. Methods: Two human ESCC cell lines EC9706 and Eca109 were selected and challenged with metformin in this study. Western blot assay was performed to detect th level of Bcl-2, Bax and Caspase-3. Scratch wound assay, transwell assay and Millicell invasion assay were used to assay the invasion and migration of EC9706 and Eca109 cells. Nude mice tumor models were used to assay the growth and lung metastasis of ESCC cells after metformin treatment. The plasma glucose level was also assayed. Results: We found that metformin significantly inhibited proliferation and induced apoptosis of both ESCC cell lines in a dose- and time-dependent manner, and the expression of Bcl-2 was down-regulated and Bax and Caspase-3 were up-regulated. Metformin significantly inhibited the invasion and migration of EC9706 and Eca109 cells (p < 0.05). mRNA and protein levels of MMP-2 and MMP-9 decreased significantly upon treatment with metformin of 10mM for 12, 24 and 48h in a time-dependent manner (p < 0.05). In line with in vitro results, in vivo experiments demonstrated that metformin inhibited tumorigenicity, inhibited lung metastasis and down-regulated the expression of MMP-2 and MMP-9. Moreover, we showed that metformin treatment did not cause significant alteration in liver and renal functions and plasma glucose level. Conclusion: Our study for the first time demonstrated the anti-invasive and anti-metastatic effects of metformin on human ESCC cells both in vitro and in vivo, which might be associated with the down-regulation of MMP-2 and MMP-9. As a whole, our results indicate the potential of metformin to be developed as a chemotherapeutic agent for patients with ESCC and might stimulate future studies on this area.

scholarly journals Synthesis and In Vitro Antitumor Activity of Novel Bivalent β-Carboline-3-carboxylic Acid Derivatives with DNA as a Potential Target

2018 ◽  
Vol 19 (10) ◽  
pp. 3179 ◽  
Author(s):  
Hongling Gu ◽  
Na Li ◽  
Jiangkun Dai ◽  
Yaxi Xi ◽  
Shijun Wang ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Cell Apoptosis ◽  
Docking Studies ◽  
In Vitro Cytotoxicity ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Dna Binding Affinity ◽  
Dose Dependent ◽  
Mcf 7

A series of novel bivalent β-carboline derivatives were designed and synthesized, and in vitro cytotoxicity, cell apoptosis, and DNA-binding affinity were evaluated. The cytotoxic results demonstrated that most bivalent β-carboline derivatives exhibited stronger cytotoxicity than the corresponding monomer against the five selected tumor cell lines (A549, SGC-7901, Hela, SMMC-7721, and MCF-7), indicating that the dimerization at the C3 position could enhance the antitumor activity of β-carbolines. Among the derivatives tested, 4B, 6i, 4D, and 6u displayed considerable cytotoxicity against A549 cell line. Furthermore, 4B, 6i, 4D, and 6u induced cell apoptosis in a dose-dependent manner, and caused cell cycle arrest at the S and G2/M phases. Moreover, the levels of cytochrome C in mitochondria, and the expressions of bcl-2 protein, decreased after treatment with β-carbolines, which indicated that 6i and 6u could induce mitochondria-mediated apoptosis. In addition, the results of UV-visible spectral, thermal denaturation, and molecular docking studies revealed that 4B, 6i, 4D, and 6u could bind to DNA mainly by intercalation.

scholarly journals Thrombospondin-1 Inhibits Angiogenesis and Promotes Follicular Atresia in a Novel in Vitro Angiogenesis Assay

Endocrinology ◽  
2010 ◽  
Vol 151 (3) ◽  
pp. 1280-1289 ◽  
Author(s):  
Samantha A. Garside ◽  
Christopher R. Harlow ◽  
Stephen G. Hillier ◽  
Hamish M. Fraser ◽  
Fiona H. Thomas
Keyword(s):  
Granulosa Cells ◽  
Caspase 3 ◽  
Polycystic Ovary ◽  
Dependent Manner ◽  
Ovary Syndrome ◽  
Angiogenesis Assay ◽  
Thrombospondin 1 ◽  
In Vitro Angiogenesis ◽  
Dose Dependent

Thrombospondin-1 (TSP-1) is a putative antiangiogenic factor, but its role in regulating physiological angiogenesis is unclear. We have developed a novel in vitro angiogenesis assay to study the effect of TSP-1 on follicular angiogenesis and development. Intact preantral/early antral follicles dissected from 21-d-old rat ovaries were cultured for 6 d in the presence or absence of TSP-1. At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. Follicles were fixed and sectioned, and follicular apoptosis was assessed by immunohistochemistry for activated caspase-3 in granulosa cells. The results showed that TSP-1 inhibited follicular angiogenesis (P &lt; 0.01) and promoted follicular apoptosis (P &lt; 0.001) in a dose-dependent manner. To determine whether the proapoptotic activity of TSP-1 is mediated by direct effects on granulosa cells, isolated granulosa cells were cultured with TSP-1 (0, 10, 100, and 1000 ng/ml) for 48 h. Apoptosis was quantified using a luminescent caspase-3/7 assay. TSP-1 promoted apoptosis of granulosa cells in a dose-dependent manner (P &lt; 0.05), suggesting that TSP-1 can act independently of the angiogenesis pathway to promote follicular apoptosis. These results show that TSP-1 can both inhibit follicular angiogenesis and directly induce apoptosis of granulosa cells. As such, it may have potential as a therapeutic for abnormal ovarian angiogenesis and could facilitate the destruction of abnormal follicles observed in polycystic ovary syndrome.

scholarly journals Induction of PI3K/Akt-Mediated Apoptosis in Osteoclasts Is a Key Approach for Buxue Tongluo Pills to Treat Osteonecrosis of the Femoral Head

2021 ◽  
Vol 12 ◽  
Author(s):  
Dan Wang ◽  
Yicheng Liu ◽  
Dandan Tang ◽  
Shujun Wei ◽  
Jiayi Sun ◽  
...  
Keyword(s):  
Femoral Head ◽  
Functional Annotation ◽  
Caspase 3 ◽  
Differentially Expressed Gene ◽  
Pathway Enrichment Analysis ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Clinical Practices ◽  
Key Genes

The Buxue Tongluo pill (BTP) is a self-made pill with the functions of nourishing blood, promoting blood circulation, dredging collaterals, and relieving pain. It consists of Angelica sinensis (Oliv.) Diels, Pheretima aspergillum (E.Perrier), Panax notoginseng (Burk.) F. H. Chen, Astragalus membranaceus (Fisch.) Bge, and Glycyrrhiza uralensis Fisch. Various clinical practices have confirmed the therapeutic effect of BTP on osteonecrosis of the femoral head (ONFH), but little attention has been paid to the study of its bioactive ingredients and related mechanisms of action. In this study, UPLC/MS-MS combined with GEO data mining was used to construct a bioactive ingredient library of BTP and a differentially expressed gene (DEG) library for ONFH. Subsequently, Cytoscape (3.7.2) software was used to analyze the protein–protein interaction between BTP and DEGs of ONFH to screen the key targets, and functional annotation analysis and pathway enrichment analysis were carried out. Finally, 34 bioactive compounds were screened, which acted on 1,232 targets. A total of 178 DEGs were collected, and 17 key genes were obtained after two screenings. By bioinformatics annotation on these key genes, a total of 354 gene ontology (GO) functional annotation analyses and 42 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were obtained. The present study found that GO and KEGG enrichment were mainly related to apoptosis, suggesting that BTP may exert an anti-ONFH effect by promoting osteoclast apoptosis. Experiments in vitro demonstrated that BTP could increase the mitochondrial membrane potential (MMP) and induce remarkable apoptosis in osteoclasts. Furthermore, we determined the apoptosis marker of cleaved(C)-caspase-3, bcl-2, and bax and found that BTP could upregulate the C-caspase-3 and bax expression in osteoclasts and decrease the expression of bcl-2, p-Akt, and p-PI3K in a dose-dependent manner, indicating that BTP could induce PI3K/Akt-mediated apoptosis in osteoclasts to treat ONFH. This study explored the pharmacodynamic basis and mechanism of BTP against ONFH from the perspective of systemic pharmacology, laying a foundation for further elucidating the therapeutic effects of BTP against ONFH.

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