Anti-Inflammatory and Anti-Osteoclastogenesis Activities of Different Kinds of Zanthoxylum bungeanum Seed Oil in Vitro.

Our previous study has exhibited that one kind of Zanthoxylum bungeanum seed oil (ZSO), extracted from Zanthoxylum bungeanum seed, had inhibitory effects on osteoclastogenesis. However, the anti-osteoclastogenesis activities of different kinds of ZSO are scarcely reported. Since inflammation is related to bone loss and osteoporosis, in this study, three kinds of ZSO, Zanthoxylum schinifolium Siebold et Zucc seed oil (ZSSO), Zanthoxylum armatum DC. seed oil (ZDSO) and Zanthoxylum bungeanum maximum seed oil (ZBSO), were obtained with Soxhlet extraction and their fatty acid constituents were detected by GC-FID. RAW264.7 macrophages induced by lipopolysaccharide (LPS) were used to evaluate the inhibitory effects of three kinds of ZSO on inflammation via detecting the expression levels of inflammatory factors by RT-qPCR. Moreover, RANKL-induced osteoclastogenesis was applied to demonstrate the anti-osteoclastogenesis activities of them through tartrate-resistant acid phosphatase (TRAP) staining and RT-qPCR. The GC-FID results exhibited that the highest constituent in ZSSO and ZDSO was oleic acid (OA) and palmitoleic acid (PLA), respectively. While linoleic acid (LA) and α-Linolenic acid (ALA) in ZBSO were dominant. At the concentration of 0.5 µL/mL, all three kinds of ZSO could decrease the expression levels of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1ß) in LPS-induced macrophages. At the concentration of 0.25 µL/mL, only ZSSO could decrease the expression levels of iNOS and COX-2, which implied the inhibitory effects of ZSSO were stronger than other ZSOs. The number of RANKL-induced osteoclasts and the expressions of nuclear factor kappa-B (NF-κB), TNF-α and IL-6 in the cells were decreased after being treated with ZSOs at the concentration of 0.5 µL/mL, while the number of RANKL-induced osteoclasts after treated with ZBSO were less than those treated with other ZSOs, this indicated that the anti-osteoclastogenesis effect of ZBSO were stronger than other ZSOs. In conclusion, the fatty acid compositions of three major kinds of ZSO were compared and the content of unsaturated fatty acids especially ω-3 polyunsaturated fatty acids in ZBSO were the highest among them. All ZSOs tested had anti-inflammatory and anti-osteoclastogenesis activities. And their anti-osteoclastogenesis effects might be related to the suppression of the NF-κB pathway.

α-Linolenic Acid Inhibits RANKL-Induced Osteoclastogenesis In Vitro and Prevents Inflammation In Vivo.

Inflammation is an important risk factor for bone-destroying diseases. Our preliminary research found that Zanthoxylum bungeanum seed oil (ZBSO) is abundant in unsaturated fatty acids and could inhibit osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. However, the key constituents in ZBSO in the prevention of osteoclastogenesis and its possible mechanism related to inflammation are still unclear. Therefore, in this study, oleic acid (OA), linoleic acid (LA), palmitoleic acid (PLA), and alpha-linolenic acid (ALA) in ZBSO, havingthe strongest effect on RANKL-induced osteoclastogenesis, were selected by a tartrate-resistant acid phosphatase (TRAP) staining method. Furthermore, the effects of the selected fatty acids on anti-inflammation and anti-osteoclastogenesis in vitro and in vivo were assessed using RT-qPCR. Among the four major unsaturated fatty acids we tested, ALA displayed the strongest inhibitory effect on osteoclastogenesis. The increased expression of free fatty acid receptor 4 (FFAR4) and ß-arrestin2 (ßarr2), as well as the decreased expression of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), nuclear factor of activated T-cells c1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) in RAW264.7 cells after ALA treatment were observed. Moreover, in ovariectomized osteoporotic rats with ALA preventive intervention, we found that the expression of TNF-α, interleukin-6 (IL-6), interleukin-1ß (IL-1ß), NFATc1, and TRAP were decreased, while with the ALA therapeutic intervention, downregulated expression of NF-κB, NFATc1, TRAP, and transforming growth factor beta-activated kinase 1 (TAK1) were noticed. These results indicate that ALA, as the major unsaturated fatty acid in ZBSO, could inhibit RANKL-induced osteoclastogenesis via the FFAR4/ßarr2 signaling pathway and could prevent inflammation, suggesting that ZBSO may be a promising potential natural product of unsaturated fatty acids and a dietary supplement for the prevention of osteoclastogenesis and inflammatory diseases.

Zanthoxylum bungeanum seed oil inhibits RANKL-induced osteoclastogenesis by suppressing ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum bungeanum Maxim (ZBM), a traditional Chinese medicine, is traditionally used for osteoporosis treatment recorded in ancient Chinese medicine work Benjingshuzheng and reported to have the anti-bone loss activity in recent studies. However, the anti-osteoporotic activities of the seed of ZBM have not been elucidated yet. Our previous study found that Zanthoxylum bungeanum Maxim seed oil (ZBSO) was rich in polyunsaturated fatty acids (PUFAs), which were reported to prevent bone loss. Thus, we propose a hypothesis that ZBSO could be a potential natural resource for anti-bone loss. AIM OF THE STUDY: To investigate whether ZBSO could prevent bone loss by targeting osteoclastogenesis and investigate the potential mechanisms in receptor-activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. MATERIALS AND METHODS: RAW264.7 cells were treated with RANKL in the presence or absence of ZBSO. The effect of ZBSO on osteoclast differentiation and bone resorption activity of RAW264.7 cells were evaluated by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, and bone resorption assay. Differentially expression genes (DEGs) and relevant pathways of different cell groups were obtained from RNA sequencing and protein-protein interaction (PPI) network analysis followed by KEGG pathway enrichment analysis. The effect of ZBSO on the RANKL-induced cell cycle change was analyzed by flow cytometry assay, and the expression of genes and proteins related to the selected pathways was further verified by RT-qPCR and western blot analysis. RESULTS: The inhibitory effects of ZBSO on osteoclast differentiation and bone resorption activity in a dose-dependent manner were demonstrated by TRAP staining, F-actin ring staining, and bone resorption assay in RANKL-induced RAW264.7 cells. Osteoclast differentiation and cell cycle pathways were the most enriched pathways based on DEGs enrichment analysis among different cell groups. The reversion effect of ZBSO on the RANKL-induced RAW264.7 cell cycle arrest at the G1 phase was observed by flow cytometry assay. Western blot results showed that ZBSO markedly decreased RANKL-induced activation of ERK, as well as the phosphorylation of c-JUN and NFATc1 expression, and subsequently suppressed osteoclast-specific genes, such as Ctsk, Trap, and Dc-stamp. CONCLUSIONS: ZBSO exhibited an inhibitory effect on osteoclastogenesis via suppressing the ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest induced by RANKL, suggesting that ZBSO may serve as a promising agent for anti-bone loss.

Study on Sentinel Lymph Node and Its Lymphatic Drainage Pattern of Breast Cancer by Contrast-Enhanced Ultrasound.

OBJECTIVES: Sentinel lymph node (SLN) and its lymphatic drainage pattern (LDP) of breast cancer were studied by contrast-enhanced ultrasound (CEUS). METHODS: From July 2017 to December 2019, patients with SLN localization of breast cancer in Sichuan Academy of Medical Sciences·Sichuan Provincial People's Hospital were selected. The sentinel lymph system of breast cancer was observed by CEUS before both operation and blue staining in the surgery. The location, number, and route of sentinel lymphatic channel (SLC) were recorded, along with the number, size, and the depth from skin of SLN. LDPs were summarized according to these basic characteristics of SLC and SLN. RESULTS: A total of 368 cases were included; 465 SLCs and 423 SLNs were detected. Most of the SLCs were originated from the outer upper quadrant of areola. Eleven LDPs were found, including 31 subtypes of LDPs. There were 6 cases of type A (1.63%), 15 cases of type B (4.08%), 223 cases of type C (57.88%), 38 cases of type D (10.33%), 2 cases of type E (0.54%), 3 cases of type F (0.82%), 50 cases of type G (13.59%), 30 cases of type H (8.15%), 2 cases of type I (0.54%), 6 cases of type J (1.63%), and 3 cases of type K (0.82%). CONCLUSIONS: The most common LDP of breast cancer was one SLC originated from the upper quadrant of areola with one SLN. CEUS can identify the LDP before surgery to reduce the false negative rate of SLN biopsy.

Cytoprotective Effect of Ligustrum robustum Polyphenol Extract against Hydrogen Peroxide-Induced Oxidative Stress via Nrf2 Signaling Pathway in Caco-2 Cells.

Ligustrum robustum is a traditional herbal tea that is widely distributed in southwest China. The health effects of L. robustum are characteristics of clearing heat, antioxidant, inducing resurgence, and improving digestion. However, the molecular mechanisms related to these effects, particularly the antioxidant mechanism, have been seldom reported. The objective of this study was to assess antioxidative capacity of L. robustum, and its protective effects and mechanisms against hydrogen peroxide (H2O2) - induced toxicity in Caco-2 cells. Total phenolic contents, free radical scavenging activity, and reducing capacity of L. robustum were measured. The effects of L. robustum on the cell viability and antioxidant defense system were explored. The expression of nuclear factor E2 related factor 2 (Nrf2) and antioxidant genes: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and glutamate cysteine ligase (GCL) were analyzed by western blot and qPCR. Pretreatment of L. robustum could significantly reduce H2O2-induced toxicity, decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA), and increase the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR). By activating the expression of Nrf2 and antioxidant genes (NQO1, HO-1, and GCL), L. robustum exerts cytoprotective effect in Caco-2 cells dealt with H2O2. Therefore, the well-established model of Caco-2 cells demonstrates that L. robustum may modulate the cytoprotective effect against the H2O2-induced oxidative stress through the Nrf2 signaling pathway.

Anticancer activities of Zanthoxylum bungeanum seed oil on malignant melanoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum bungeanum Maxim. (ZBM), a Chinese herb medicine and food additive, has been shown to have broad-spectrum beneficial effects. However, the anticancer activities of its seed have not been reported. AIM OF THE STUDY: for the first time investigated the anti-proliferation activity of seed oil of ZBM (ZBSO) on melanoma A375 cells as well as the underlying mechanisms. MATERIALS AND METHODS: The chemical composition of ZBSO was analyzed by Ultra Performance Liquid Chromatography. A375 cells exposure at different concentrations of ZBSO to examine the selectivity versus normal skin cells, invasion, apoptosis and cell cycle arrest. Furthermore, transcriptome analysis was employed to investigate potential anticancer mechanisms of ZBSO. RESULTS: Major compounds of ZBSO were identified and unsaturated fatty acid made up the major compound. ZBSO-treated A375 cells showed more typical apoptotic morphologic features than normal cells. ZBSO can significantly inhibit invasion and proliferation of A375 cells by G1 phase arrest and induction of apoptosis. Transcriptome analysis showed that ZBSO may affect cell cycle and MAPK signaling pathway of A375 cells. CONCLUSION: ZBSO possessed anticancer activities that were selectively effective to A375 cells. This study support the hypothesis that ZBSO is a capable candidate for anti-melanoma agent, and provide new insights for future work on investigating the utilization of ZBSO in malignant melanoma treatment.

Differential gene expression profiles in paclitaxel-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells.

AIM: To elucidate the molecular mechanism of cell cycle arrest and apoptosis of MCF-7 cells induced by paclitaxel. METHODS: Flow cytometry was used to determine the cell cycle changes of MCF-7 cells upon paclitaxel treatment. Gene expression profiles of MCF-7 cells induced by paclitaxel were obtained by using cDNA microarrays containing 9984 genes and expressed sequence tags (ESTs). RESULTS: Cell cycle analysis showed that 77.8% of cells arrested at G2/M phase and 1.3% of cells underwent apoptosis upon 100 nmol x L(-1) paclitaxel treatment for 24 hours; cDNA microarray results revealed that 27 and 77 genes were differentially expressed upon 12.5 nmol x L(-1) (IC50) and 100 nmol x L(-1) paclitaxel treatment, respectively. CONCLUSION: Paclitaxel stabilized microtubules and caused G2/M cell cycle arrest and apoptotic cell death in a concentration-dependent manner, which is associated with the regulation of selected genes related to microtubule assembly and cytoskeleton, cell cycle regulation, and DNA repair and apoptosis.