Chlorogenic acid suppresses miR-460a in the regulation of Bcl-2, causing interleukin-1ß reduction in thiram exposed chondrocytes via caspase-3/caspase-7 pathway.

BACKGROUND: Apoptosis is thought to be involved in all processes, including normal cell cycle, immune system, atrophy, embryonic development, and chemical-induced cellular damage. However, if the normal apoptotic process fails, the results might be disastrous, e.g., chondrocytes damage in tibial dyschondroplasia (TD). TD is a worldwide issue in the poultry sector due to thiram toxicity. Thiram (Tetramethyl thiuram disulfide) is a dithiocarbamate pesticide and fungicide commonly used in horticulture to treat grains meant for seed protection and preservation. PURPOSE: According to prior studies, chlorogenic acid (CGA) is becoming essential for regulating apoptosis. But still, the specific role of CGA in chondrocyte cells remains unclear. The present study explored the molecular mechanism of CGA on chondrocytes' apoptosis with B-cell lymphoma 2 signaling under the effect of miR-460a. METHODS: An in vivo and in vitro study was performed according to our previously developed methodology. Flow cytometry, western blotting, reverse transcription-quantitative polymerase chain reaction, and immunofluorescence assay were used to investigate the involvement of apoptosis and inflammasome related pathways. RESULTS: The CGA decreased the apoptosis rate with the deactivation of miR-460a, accompanied by the activation of Bcl-2. The high expression of miR-460a reduced the cell viability of chondrocytes in vitro and in vivo, that led to the interleukin-1ß production. While the apoptotic executioners (caspase-3 and caspase-7) acted upstream in miR-460a overexpressing cells, and its depletion downgraded these executioners. The CGA administrated cells negatively regulated miR-460a expression and thus indicating the deactivation of the apoptotic and inflammasome related pathways. CONCLUSION: Chlorogenic acid had a negative effect on miR-460a, setting off specific feedback to regulate apoptotic and inflammasome pathways, which might be a key feature for chondrocytes' survival.

Chlorogenic acid suppresses mitochondrial apoptotic effectors Bax/Bak to counteract Nod-like receptor pyrin domain 3 (NLRP3) inflammasome in thiram exposed chondrocytes.

BACKGROUND: Tibial dyschondroplasia (TD) is a common disease characterized by proliferation and the deterioration of growth plate's chondrocytes due to widespread utilization of thiram in the agriculture and industrial sector. PURPOSE: In recent years, Nod-like receptor pyrin domain 3 (NLRP3) inflammasome has become a dilemma in the occurrence of many diseases. According to many research investigations, NLRP3 inflammasome has been linked to various diseases caused by pesticides and environmental toxins. Its involvement in such conditions opens up new treatment approaches. However, the role of the NLRP3 inflammasome in the development of TD is not fully understood under the impact of chlorogenic acid (CGA). METHODS: Chondrocytes were cultured with our previously developed methodology from growth plates. After morphological and molecular identification, chondrocytes were split into different groups to investigate the efficacy of chlorogenic acid. Cell apoptosis was determined through flow cytometry and Tunnel assay. Furthermore, RT-qPCR, immunofluorescence, and western blotting techniques were used to check marker genes and proteins expression. RESULTS: In thiram-induced TD, Bax/Bak activation persuade a parallel pathway, mediated by the NLRP3 base inflammasome. It is worth mentioning that the apoptotic executioners (caspase-3 and caspase-7) act upstream for inflammasome. Furthermore, chondrocytes' ability to undergo mitochondrial apoptosis was governed by anti-apoptotic members, e.g., Bcl-2 and Bcl-xl. Equilibrium of these anti-apoptotic proteins ensured appropriate regulation of apoptosis during the development and survival of chondrocytes. CONCLUSION: Chondrocytes have ability to undergo Bax/Bak-mediated apoptosis and generate pro-inflammatory signals, e.g., NLRP3 in thiram-induced TD. So, the Nod-like receptor pyrin domain 3 is the potential target to eliminate TD at all stages of pathology, while drugs, e.g., CGA, can significantly improve chondrocytes' survival by targeting these pro-inflammatory signals.

Five new C17/C15 sesquiterpene lactone dimers from Carpesium abrotanoides.

Five new unusual C17/C15 sesquiterpene lactone dimers, carabrodilactones A-E (1-5), along with four known common C15/C15 SLDs, carpedilactones A and B (6 and 7), faberidilactone A (8), and faberidilactone C (9), were isolated from the whole plants of Carpesium abrotanoides. The structures of 1-5 featured a flexible C-11/C-13' linked single bond between two sesquiterpene units and a tailed acetyl connected to the C-13 position. The preferential conformation of 1-5 was elucidated by the diagnostic NMR data of geminal proton of H-13. The biogenetic pathway of 1-5 was proposed to involve Stetter and Michael addition reactions. In addition, compound 1 exhibited significant cytotoxicities against the four cell lines (A549, HCT116, MDA-MB, and BEL7404 cells) with IC50 value in the range of 3.08-8.05 µM, while compounds 2-5 showed weak cytotoxicities.

E. adenophorum induces Cell Cycle Arrest and Apoptosis of Splenocytes through the Mitochondrial Pathway and Caspase Activation in Saanen Goats.

The precise cytotoxicity of E. Adenophorum in relation to the cell cycle and apoptosis of splenocytes in Saanen goats remains unclear. In the present study, 16 Saanen goats were randomly divided into four groups, which were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The results of TUNEL, DAPI and AO/EB staining, flow cytometry analysis and DNA fragmentation assays showed that E. adenophorum induced typical apoptotic features in splenocytes, suppressed splenocyte viability, and caused cell cycle arrest in a dose-dependent manner. However, westernblot, ELISA, qRT-PCR and caspase activity analyses showed that E. adenophoruminhibited Bcl-2 expression, promoted Bax translocation to the mitochondria, triggered the release of Cytc from the mitochondria into the cytosol, and activated caspase-9 and -3 and the subsequent cleavage of PARP. Moreover, in E. adenophorum-induced apoptosis, the protein levels of Fas, Bid, FasL and caspase-8 showed no significant changes. E. adenophorum treatment induced the collapse of ΔΨm. Moreover, these data suggested that E. adenophorum induces splenocyte apoptosis via the activation of the mitochondrial apoptosis pathway in splenocytes. These findings provide new insights into the mechanisms underlying the effects of E. adenophorum cytotoxicity on splenocytes.

Induction and mechanism of HeLa cell apoptosis by 9-oxo­10, 11-dehydroageraphorone from Eupatorium adenophorum.

9-Oxo-10, 11-dehydroageraphorone (euptox A), a cadenine sesquiterpene, is the main toxin from Eupatorium adenophorum. The aim of the present study was to examine the induction and mechanism of HeLa cell apoptosis by euptox A. The apoptosis­inducing effect of the euptox A on HeLa cells was examined by MTT assay. The underlying mechanism was analyzed by flow cytometry and quantitative PCR. Flow cytometry results suggested that euptox A effectively inhibited the proliferation of HeLa cells, arrested the cell cycle transition from S to G2/M phase, did not continue to complete the cell cycle activity (mainly from 4 times and mitosis), and induced cell proliferation. The RT-qPCR detection results showed that euptox A induced apoptosis by improving the gene expression level of apoptotic proteases such as caspase-10 in HeLa cells. Its mechanism of action was associated with the upregulation of apoptotic gene expression and arresting of the cell cycle.

Clinical efficacy of 9-oxo-10, 11-dehydroageraphorone extracted from Eupatorium adenophorum against Psoroptes cuniculi in rabbits.

BACKGROUND: Animal acariasis is one of the important veterinary skin diseases. Chemical drugs have been widely used to treat and control this kind of disease. But many chemicals control could increase resistance in target species, toxicity and environmental hazards. We found that the 9-oxo-10, 11-dehydroageraphorone (euptox A) extracted from E. adenophorum has strong toxicity against P. cuniculi in vitro, but the in vivo acaricidal actions of euptox A have yet to be investigated. RESULTS: A 14-day experiment was performed using rabbits that were naturally infested with P. cuniculi on a farm. Rabbits were randomly divided into five groups; animals in groups A, B and C were treated in each ear topically with 4.0 ml of 2.0 and 1.0 g/L (w/v) euptox A, respectively. Animals in groups D and E were treated with ivermectin (by injection; positive controls) and glycerol with water only (by embrocation; negative controls), respectively. Each rabbit was treated twice with separate treatments on days 0 and 7. Rabbits were observed daily and detailed examinations were performed on days 0, 7 and 14, to inspect the presence or absence of mites and scabs/crusts. Seven days after the initial treatment, the mean clinical scores (presence of scabs/crusts) decreased from 3.48, 3.37, 3.43 and 3.45 to 0.37, 0.42, 0.78 and 0.38 in the ears of animals in groups A, B , C and D, respectively, which were similar to the observations recorded in the positive control rabbits. However, the clinical score for negative control rabbits did not increase significantly (P > 0.05) during the experiment, and this changed from 3.32 to 3.37 in the ears, and there were no significant differences in clinical efficacy between left and right ears. After two treatments (0 and 7 d), the rabbits in groups A, B, C and D had recovered completely 14 days after the last treatment and no recurrences of infection were observed. CONCLUSIONS: These results indicate that euptox A was potent compounds for the effective control of animal P. cuniculi in vivo.

Acaricidal activity of 9-oxo-10,11-dehydroageraphorone extracted from Eupatorium adenophorum in vitro.

The acaricidal activity of the 9-oxo-10,11-dehydroageraphorone (euptox A), a cadenine sesquiterpene from Eupatorium adenophorum (E. adenophorum) against Sarcoptes scabiei and Psoroptes cuniculi was tested in vitro. A complementary log-log (CLL) model was used to analyze the data of the toxicity tests in vitro. The results showed euptox A had strong toxicity against mites, killing all S. scabiei at 3 and 4 mg/ml (m/v) concentration, while 4 mg/ml euptox A was also found to kill all P. cuniculi within a 4 h period. Similarly, 2, 3 and 4 mg/ml concentration of euptox A had strong toxicity against S. scabiei, with median lethal time (LT50) values at 0.687, 0.526, 0.326 h, respectively. 3 mg/ml and 4 mg/ml showed strong acaricidal action against P. cuniculi; the LT50 values were 0.693 and 0.493 h, respectively. The median lethal concentration (LC50) values were 1.068 mg/ml for Scabies mite and 0.902 mg/ml for P. cuniculi in 2 h. The results indicate that euptox A has strong acaricidal activity and may exploit as novel drugs for the effective control of animal acariasis.

[Study on extraction and purification process of total ginsenosides from Radix Ginseng].

OBJECTIVE: To optimize the technological parameters of the extraction and purification process of total ginsenosides from Radix Ginseng. METHODS: With the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1, the orthogonal design was adopted to optimize the extraction process. The purification process was studied by optimizing the elutive ratio of total ginsenosides as the marker. HPLC and spectrophotometer were employed for the study. RESULTS: The optimum conditions were as follows:Using 8 times volume of 75% ethanol extracting for 120 minutes and 2 times, the extraction temperature was 85 degrees C. AB-8 macroporous resin was selected, and the eluant was 4 BV 70% ethanol. CONCLUSION: The optimal conditions of extracting and purifying the total ginsenosides from Radix Ginseng is feasible.