Caspase-3 Protein Expression Research Articles

Quantum Molecular Resonance Inhibits NLRP3 Inflammasome/Nitrosative Stress and Promotes M1 to M2 Macrophage Polarization: Potential Therapeutic Effect in Osteoarthritis Model In Vitro.

This study aimed to investigate the anti-inflammatory effects of Quantum Molecular Resonance (QMR) technology in an in vitro model of osteoarthritis-related inflammation. The study used THP-1-derived macrophages stimulated with lipopolysaccharide and hyaluronic acid fragments to induce the expression of inflammatory cytokines and nitrosative stress. QMR treatment inhibited COX-2 and iNOS protein expression and activity and reduced NF-κB activity. Furthermore, QMR treatment led to a significant reduction in peroxynitrite levels, reactive nitrogen species that can form during inflammatory conditions, and restored tyrosine nitration values to those similar to sham-exposed control cells. We also investigated the effect of QMR treatment on inflammasome activation and macrophage polarization in THP-1-derived macrophages. Results showed that QMR treatment significantly decreased NLRP3 and activated caspase-1 protein expression levels and downregulated IL-18 and IL-1β protein expression and secretion. Finally, our findings indicate that QMR treatment induces a switch in macrophage polarization from the M1 phenotype to the M2 phenotype.

LncRNA DRAIC regulates the proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells by targeting let-7i-5p

Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration.

To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Spermidine alleviating oxidative stress and apoptosis by inducing autophagy of granulosa cells in Sichuan white geese

Spermidine have been reported a role in antioxidative, antiaging, and antiinflammatory. Oxidative stress causes granulosa cell (GC) apoptosis, follicular atresia, and impairs poultry reproductive functions. Studies have found that autophagy is the protective mechanism against antioxidant stress and apoptosis in cells. However, the relationship between spermidine-induced autophagy, oxidative stress, and apoptosis in goose GCs remains unclear. In this study, we investigated the autophagy mechanism to mediate spermidine effects on the alleviation of oxidative stress and apoptosis in goose GCs. Follicular GCs were treated with spermidine combination with 3-Nitropropanoic acid (3-NPA), rapamycin (RAPA), and chloroquine (CQ) or with hydrogen peroxide, RAPA, and CQ. Spermidine upregulated the ratio of LC3-II/I, inhibited the accumulation of p62 protein, and induced autophagy. 3-NPA treatment significantly increased ROS production, MDA content, SOD activity, cleaved CASPASE-3 protein expression, and decreased BCL-2 protein expression in follicular GCs. Spermidine inhibited oxidative stress and apoptosis induced by 3-NPA. In addition, hydrogen peroxide-induced oxidative stress was inhibited by spermidine. However, the inhibitory effect of spermidine was eliminated under chloroquine. Our results demonstrated that spermidine relieved oxidative stress and apoptosis of GCs by inducing autophagy, indicating that spermidine has a great potential to maintain proteostasis and sustain granulosa cell viability in geese.

The Protective Effect of Nutraceuticals on Hepatic Ischemia-Reperfusion Injury in Wistar Rats.

Nutraceuticals are bioactive compounds present in foods, utilized to ameliorate health, prevent diseases, and support the proper functioning of the human body. They have gained attention due to their ability to hit multiple targets and act as antioxidants, anti-inflammatory agents, and modulators of immune response and cell death. Therefore, nutraceuticals are being studied to prevent and treat liver ischemia-reperfusion injury (IRI). This study evaluated the effect of a nutraceutical solution formed by resveratrol, quercetin, omega-3 fatty acid, selenium, ginger, avocado, leucine, and niacin on liver IRI. IRI was performed with 60 min of ischemia and 4 h of reperfusion in male Wistar rats. Afterward, the animals were euthanized to study hepatocellular injury, cytokines, oxidative stress, gene expression of apoptosis-related genes, TNF-α and caspase-3 proteins, and histology. Our results show that the nutraceutical solution was able to decrease apoptosis and histologic injury. The suggested mechanisms of action are a reduction in gene expression and the caspase-3 protein and a reduction in the TNF-α protein in liver tissue. The nutraceutical solution was unable to decrease transaminases and cytokines. These findings suggest that the nutraceuticals used favored the protection of hepatocytes, and their combination represents a promising therapeutic proposal against liver IRI.

Polysaccharide extracted from the Sargassum fusiforme induces cell cycle arrest and apoptosis of B16F10 melanoma cells through the PI3K/AKT pathway.

SARGASSUM FUSIFORME: (S. fusiforme) is a brown alga that has been utilized as a medicine for a long time. Polysaccharides extracted from S. fusiforme demonstrate antitumor activities. The impact of S. fusiforme polysaccharides (SFPS 191,212) on the proliferation, apoptosis, and cell cycle kinetics of B16F10 murine melanoma cells were thoroughly investigated in this work. The anticancer activities of the SFPS 191,212 compounds were assayed in the B16F10 cells at both transcriptional and translational levels. The compound exhibited concentration-dependent effects. Moreover, SPFS 191,212 increased the numbers of apoptotic cells and arrested the cell cycle in the S phase of the quantitative real-time PCR. From western blotting, it was verified that the SFPS 191,212 treatment improved the expression of Bax, Caspase-9, and Caspase-3 genes and proteins, while it reduced phosphatidylinositol 3 kinase and Bcl-2 genes and proteins, suggesting the involvement of mitochondria. Overall, SFPS 191,212 can be further explored as a potential functional food or adjuvant agent for the prevention or treatment of melanoma.

Gastroprotective Effects of the Aqueous Extract of Finger Citron Pickled Products against Ethanol-Induced Gastric Damage: In Vitro and In Vivo Studies.

Finger citron pickled products (FCPP), as folk remedies, are famous in southern China for protecting gastric mucosa. However, the gastric mucosa protection of FCPP has not been reported yet, and its effective mechanism is unclear. In this study, the protective mechanism of FCPP aqueous extract on gastric mucosa was investigated in vitro and in vivo for the first time, using human gastric mucosa epithelial cells (GES-1) and acute alcoholic gastric ulcer rat model respectively. Furthermore, we also investigated the main substances in the aqueous extract that exert gastroprotective activity using a GES-1 scratch test and basic chemical composition analysis. FCPP aqueous extract was found to play a protective and reparative role in GES-1 by promoting the secretion of trefoil factor thyroid transcription factor 2 (TFF2) and inhibiting the secretion of tumor necrosis factor-α (TNF-α) in cells damaged by alcohol. The ulcer index of gastric tissue induced by alcohol was significantly decreased (p < 0.01) after pretreatment with FCPP aqueous extract, indicating that FCPP aqueous extract had a good protective effect on the stomach mucosa. Moreover, FCPP aqueous extract could increase superoxide dismutase (SOD) activity and inhibit malondialdehyde (MDA) content, exhibiting good antioxidant capacity. Aqueous extract of FCPP could also effectively inhibit the increase of cytokines TNF-α, interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum of rats, and promote the increase of anti-inflammatory cytokines interleukin-10 (IL-10) to some extent. Furthermore, FCPP aqueous extract could inhibit the expression of nuclear factor kappa-B (NF-κB/P65) protein, caspase-1 protein and IL-1β protein in the gastric tissue of rats, while promoting the expression of IκBα protein, indicating that the gastric mucosa protection effects of FCPP aqueous extract were mainly dependent on the NF-κB/caspase-1/IL-1β axis. The polysaccharides in FCPP aqueous extract might be the main components that exerted gastroprotective activity, as demonstrated by GES-1 cell scratch assay. This study confirmed that FCPP aqueous extract presented promising potential in protecting gastric mucosa and avoiding gastric ulcers, which could provide an experimental basis for further utilizing the medicinal value and developing new products of FCPP.

Danshensu inhibits Aβ aggregation and neurotoxicity as one of the main prominent features of Alzheimer's disease

It has been found that the main cause of neurodegenerative proteinopathies, especially Alzheimer's disease (AD) is the formation of Aβ amyloid plaques, which can be regulated by application of potential small molecules. In the present study, we aimed to investigate the inhibitory effect of danshensu on Aβ(1-42) aggregation and relevant apoptotic pathway in neurons. A broad range of spectroscopic, theoretical, and cellular assays were done to investigate the anti-amyloidogenic characteristics of danshensu. It was found that danshensu triggers its inhibitory effect against Aβ(1-42) aggregation through modulation of hydrophobic patches as well as structural and morphological changes through a stacking interaction. Furthermore, it was observed that incubation of Aβ(1-42) samples with danshensu during aggregation process recovered the cell viability and mitigated the expression of caspase-3 mRNA and protein as well caspase-3 activity deregulated by Aβ(1-42) amyloid fibrils alone. In general, obtained data showed that danshensu potentially inhibits Aβ(1-42) aggregation and associated proteinopathies through regulation of apoptotic pathway in a concentration-dependent manner. Therefore, danshensu may be used as a promising biomolecule against the Aβ aggregation and associated proteinopathies, which can be further analyzed in the future studies for the treatment of AD.

The synbiotic mixture of lactulose and Bacillus coagulans protects intestinal barrier dysfunction and apoptosis in weaned piglets challenged with lipopolysaccharide

BackgroundLactulose as an effective prebiotic protects intestinal mucosal injury. Bacillus coagulans is widely used in feed additives because of its ability to promote intestinal health. Our previous study suggests that the combination of lactulose and Bacillus coagulans may be a good candidate as alternative for antibiotic growth promoters. However, the in vivo effects of lactulose and Bacilluscoagulans on growth and intestinal health under immune challenge in piglets remains unclear. The objective of this study is to explore the protective effects of synbiotic containing lactulose and Bacillus coagulans on the intestinal mucosal injury and barrier dysfunction under immune challenge in weaned piglets.MethodsTwenty four weaned piglets were assigned to 4 groups. Piglets in the CON-saline and LPS-LPS group were fed the basal diet, while others were fed either with chlortetracycline (CTC) or synbiotic mixture of lactulose and Bacillus coagulans for 32 d before injection of saline or lipopolysaccharide (LPS). Piglets were sacrificed 4 h after LPS injection to collect samples to determine intestinal morphology, integrity and barrier functions as well as relative genes and proteins.ResultsOur data showed that no differences were observed in the growth performance of the four test groups. LPS injection induced higher serum diamine oxidase activities, D-lactic acid levels, and endotoxin status, lower villus height and ratio of villus height to crypt depth, greater mRNA and lower protein expression related tight junction in both jejunum and ileum. In addition, a higher apoptosis index, and protein expression of Bax and caspase-3 were also observed in the LPS challenge group. Interestingly, dietary synbiotic mixture with lactulose and Bacillus coagulans protected against LPS-induced intestinal damage, barrier dysfunction and higher apoptosis as well as CTC.ConclusionsOur data suggest that dietary supplementation of synbiotic mixture with lactulose and Bacillus coagulans showed resilience to LPS-induced intestinal morphological damage, barrier dysfunction and aggressive apoptosis in piglets as well as the protective effects of CTC. These results indicate that synbiotic mixture of lactulose and Bacillus coagulans showed beneficial effects on performance and resilience to acute immune stress in weaned piglets.

Protective effect of epalrestat on peripheral nerves in rats with diabetic peripheral neuropathy via NF κB pathway

Purpose: To study the protective effect of epalrestat on peripheral nerves in rats with diabetic peripheral neuropathy (DPN).&#x0D; Methods: A total of 36 Sprague-Dawley rats were randomly divided into normal, model, and epalrestat groups, each containing 12 rats. The morphology of the neurons was assessed using hematoxylin-eosin (H&amp;E) staining. The expressions of B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined via immunohistochemistry. The relative protein expressions of NF-κB and Caspase3 were determined via Western blotting, and apoptosis was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.&#x0D; Results: The morphology of the neurons was clear and intact in the normal group, disordered and destroyed in the model group, and was improved in the epalrestat group. The other two groups had a significantly higher positive expression of Bax and a significantly lower positive expression of Bcl-2 than the normal group (p &lt; 0.05), while the epalrestat group had a significantly lower positive expression of Bax, and a significantly higher positive expression of Bcl-2 than the model group (p &lt; 0.05). Furthermore, the protein expressions of NF-κB and Caspase3 were increased in other groups compared with normal group (p &lt; 0.05), while they declined in epalrestat group compared with model group (p &lt; 0.05). The apoptosis rate was significantly lower in epalrestat group than in model group (p &lt; 0.05).&#x0D; Conclusion: Epalrestat inhibits neuronal apoptosis by suppressing the NF-κB signaling pathway, thereby exerting a neuroprotective effect in DPN rats. Further studies would be required to validate the molecular mechanism.

A combined network pharmacology and molecular biology approach to investigate the potential mechanisms of G-M6 on ovarian cancer

Ginsenoside 3β,12β,21α,22β-Hydroxy-24-norolean-12-ene (G-M6), a phase I metabolite of anti-tumor medication 20(R)-25-methoxyl-dammarane-3β,12β,20-triol (AD-1), beats the parent drug in anti-ovarian cancer efficacy. The mechanism of action for ovarian cancer, however, is uncertain. Using network pharmacology, human ovarian cancer cells and nude mouse ovarian cancer xenotransplantation model, the anti-ovarian cancer mechanism of G-M6 was preliminarily explored in this study. The PPAR signal pathway is the key signal pathway of the G-M6 anti-ovarian cancer mechanism, according to data mining and network analysis. Docking tests demonstrated that the bioactive chemical G-M6 was capable of forming a stable bond with the PPARγ target protein capsule. Using human ovarian cancer cells and xenograft model of ovarian cancer to evaluate the anticancer activity of G-M6. The IC50 value of G-M6 was 5.83±0.36, lower than AD-1 and Gemcitabine. The tumor weight of the RSG 80 mg/kg group (C), G-M6 80 mg/kg group (I), and RSG 80 mg/kg + G-M6 80 mg/kg group (J) after the intervention was as follows: C < I < J. The tumor inhibition rates of groups C, I, and J were 28.6%, 88.7%, and 92.6%, respectively. When RSG and G-M6 are combined to treat ovarian cancer, q = 1.00 is calculated according to King's formula, which indicates that RSG and G-M6 have additive effects. Its molecular mechanism may involve the up-regulation of PPARγ and Bcl-2 protein expressions, and the down-regulation of Bax, Cytochrome C (Cyt. C), Caspase-3, and Caspase-9 protein expressions. These findings serve as a reference for further research into the processes behind ginsenoside G-M6′s ovarian cancer therapy.

Newcastle disease virus LaSota strain induces apoptosis and activates the TNFα/NF-κB pathway in canine mammary carcinoma cells.

Spontaneous canine mammary carcinomas (CMCs) have been widely considered a good research model for human breast cancers, which brings much attention to CMCs. In recent years, the oncolytic effect of Newcastle disease virus (NDV) on cancer cells has been widely studied, but its effect on CMCs is still unclear. This study aims to investigate the oncolytic effect of NDV LaSota strain on canine mammary carcinoma cell line (CMT-U27) in vivo and in vitro. The in vitro cytotoxicity and immunocytochemistry experiments showed that NDV selectively replicated in CMT-U27 cells, and inhibited cell proliferation and migration but not in MDCK cells. KEGG analysis of transcriptome sequencing indicated the importance of the TNFα and NF-κB signalling pathways in the anti-tumour effect of NDV. Subsequently, the significantly increased expression of TNFα, p65, phospho-p65, caspase-8, caspase-3 and cleaved-PARP proteins in the NDV group suggested that NDV induced CMT-U27 cells apoptosis by activating the caspase-8/caspase-3 pathway and the TNFα/NF-κB signalling pathway. Nude mice tumour-bearing experiments showed that NDV could significantly decrease the growth rate of CMC in vivo. In conclusion, our study demonstrates the effective oncolytic effects of NDV on CMT-U27 cells in vivo and in vitro, and suggests NDV as a promising candidate for oncolytic therapy.

Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation, apoptosis, and migration of human ectopic endometrial stromal cells: based on JAK2/STAT3 signaling pathway

Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P&lt;0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P&lt;0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P&lt;0.05 or P&lt;0.01), down-regulate the expression of Bcl-2(P&lt;0.01), decrease the cell migration rate(P&lt;0.05 or P&lt;0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P&lt;0.05 or P&lt;0.01), and levels of p-JAK2 and p-STAT3(P&lt;0.05 or P&lt;0.01). Compared with the SR and CR groups, CM group showed low cell viability(P&lt;0.01), high protein expression of caspase-3 and Bax(P&lt;0.05 or P&lt;0.01), and low protein expression of Bcl-2 and p-JAK2(P&lt;0.05). After incubation with CM, the apoptosis rate was higher(P&lt;0.05) and the migration rate was lower(P&lt;0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P&lt;0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.

Curcumol, a major terpenoid from Curcumae Rhizoma, attenuates human uterine leiomyoma cell development via the p38MAPK/NF-κB pathway.

Uterine fibroids (UFs) are the most common benign tumors in women of reproductive age. Curcumae Rhizoma, the main essential oil component of which is curcumol, is widely used for the treatment of phymatosis in China due to its antitumor, anti-inflammatory, antithrombin, anti-tissue fibrosis and anti-oxygen pharmacological activities, but its potential for the treatment of UFs has not been evaluated. This study aimed to investigate the effects and mechanisms of curcumol intervention in human uterine leiomyoma cells (UMCs). Putative targets of curcumol intervention in UFs were identified using network pharmacology strategies. Molecular docking was performed to assess the binding affinity of curcumol to core targets. A concentration gradient of curcumol (0, 50, 100, 200, 300, 400 and 500μM) or RU-486 (mifepristone, 0, 10, 20, 40, 50, and 100μM) was applied to UMCs, and cell viability was detected by the CCK-8 assay. Cell apoptosis and cell cycle were examined by flow cytometry, and cell migration was assessed by a wound-healing assay. Additionally, the mRNA and protein expression levels of critical pathway components were evaluated by RT‒PCR and western blotting. Finally, the actions of curcumol on different tumor cell lines were summarized. Network pharmacology predicted 62 genes with roles in the treatment of UFs with curcumol, and MAPK14 (p38MAPK) displayed a higher interaction degree. GO enrichment and KEGG analyses revealed that the core genes were abundantly enriched in the MAPK signaling pathway. The molecular binding of curcumol to core targets was relatively stable. In UMCs, 200, 300 and 400μM curcumol treatment for 24h decreased cell viability compared with that in the control group, and the greatest effect was detected at 48h and maintained until 72h. Curcumol arrested cells in the G0/G1 phase and subsequently suppressed mitosis, promoted early apoptosis and reduced the degree of wound healing in a concentration-dependent manner in UMCs. Furthermore, 200μM curcumol decreased the mRNA and protein expression of p38MAPK, the mRNA expression of NF-κB, and the protein expression of Ki-67 and increased the mRNA and protein expression of Caspase 9. Curcumol (300 and 400μM) decreased the mRNA and protein expression of p38MAPK, NF-κB, and Ki-67 and increased the protein expression of Caspase 9 in UMCs. Curcumol was demonstrated to treat tumor cell lines, including breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer and nasopharyngeal carcinoma, but its effects on benign tumors have not yet been reported. Curcumol suppresses cell proliferation and cell migration while arresting the cell cycle in the G0/G1 phase and inducing cell apoptosis in UMCs via a mechanism related to p38MAPK/NF-κB pathway regulation. Curcumol may be a potential therapeutic and preventive agent in the treatment of benign tumors such as UFs.

Anti-TLR4 IgG2 Prevents Acetaminophen-induced Acute Liver Injury through the Toll-like Receptor 4/MAPKs Signaling Pathway in Mice.

Acetaminophen (APAP) is a widely used antipyretic and analgesic. If taken in excess, it can cause severe drug-induced acute liver injury. The purpose of this study was to investigate the effects of anti-TLR4 IgG2 on APAP-induced liver injury and its underlying mechanisms. We injected APAP into the abdominal cavity of mice to establish a liver injury model. Mice were divided into the control group, APAP group, and APAP + anti-TLR4 IgG2 group. In order to verify the implication of the toll-like receptor4 and mitogen-activated protein kinases activation (TLR4/MAPKs) signaling pathway, mice were intraperitoneally injected with a TLR4 / MAPKs inhibitor anti-TLR4 IgG2. We evaluated the effects of TLR4 IgG2 on the antioxidant, anti-apoptotic, anti-inflammatory, and liver histopathology of APAP mice. In addition, the expression of the TLR4 / MAPKs signaling pathway was detected by Western blot. Our study showed that APAP mouse models were successfully established; however, pretreatment with anti-TLR4 IgG2 alleviated APAP-induced hepatic injury, as evidenced by the 24-h survival rate. Meanwhile, anti-TLR4 IgG2 prevented the elevation of serum biochemical parameters and lipid profile. Furthermore, compared with the APAP group, hepatic antioxidants, including 3- Nitrotyrosine, high mobility group protein B1, superoxide dismutase, catalase, and glutathione, were increased in APAP + anti-TLR4 IgG2 group. In contrast, a significant decrease was observed in the levels of the malondialdehyde, which is a lipid peroxidation product. Moreover, the western blotting analysis showed that anti-TLR4 IgG2 treatment inhibited the activation of the apoptotic pathway by increasing Bcl-2 and decreasing Bax, P53, and cleaving caspase-3 / caspase-3 protein expression. These results were further validated by TUNEL staining and immunohistochemical. Histopathological observation also revealed that pretreat-ment with anti-TLR4 IgG2 could significantly reverse hepatocyte inflammatory infiltration, congestion, and necrosis in liver tissues by APAP. Importantly, anti-TLR4 IgG2 effectively alleviated APAP-induced liver injury by inhibiting tolllike receptor4 and mitogen-activated protein kinases activation signaling pathways (TLR4/MAPKs). The results clearly suggest that the underlying molecular mechanisms in the hepatoprotection of anti-TLR4 IgG2 in APAP-induced hepatotoxicity may be due to its antioxidation, anti-apoptosis, and anti-inflammation effects through inhibition of the TLR4/MAPKs signaling axis.

Expression of MiR-20a-5p and its target gene in colon cancer and its effect on the proliferation and apoptosis of colon cancer cells.

Abstract. We investigated the expression of micro ribonucleic acid (miR)-20a-5p and its target gene, breast cancer metastasis suppressor 1 like (BRMS1L), in colon cancer tissues and their effects on the proliferation and apoptosis of colon cancer cells. The dual luciferase assay was used to detect the targeted regulation of miR-20a-5p on BRMS1L. The expression levels of miR-20a-5p and BRMS1L in colon cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). MiR-20a-5p mimic and mimic negative control (NC) were transfected into the colon cancer cell line SW480 by the liposome transient transfection method. The MTT assay, monoclonal formation of cancer cells, and flow cytometry were used to detect cell proliferation and apoptosis. The expres-sion level of miR-20a-5p in colon cancer tissues was significantly higher than that in adjacent tissues, and the expression level of BRMS1L was significantly lower than that in adjacent tissues. The expression level of miR-20a-5p was significantly correlated with tumor-node-metastasis (TNM) stage, lymph node metastasis, in-vasion depth, and differentiation degree. The higher the expression level of miR-20a-5p, the more advanced the TNM stage and invasion depth, and the easier it is for lymph nodes to metastasize (p&lt;0.05). Compared with the control and the miR-NC groups, the miR-20a-5p group’s cell proliferation ability, expression of CyclinD1 and B-cell lymphoma-2 (Bcl-2) were significantly increased, while apoptosis ability and caspase-3 protein expression were significantly decreased (p&lt;0.05). The expression of miR-20a-5p in colon cancer tissues and cells in-creased. Overexpression of miR-20a-5p could promote the proliferation of colon cancer cells and inhibit their apoptosis.

Effect of Wenyang Zhenshuai Granules on autophagy and apoptosis of myocardial cells in septic rats via regulating miR-132-3p/UCP2 expression

This study aimed to investigate the effect of Wenyang Zhenshuai Granules(WYZSG) on autophagy and apoptosis of myocardial cells in rats with sepsis via regulating the expression of microRNA-132-3p(miR-132-3p)/uncoupling protein 2(UCP2). Sixty SD rats were randomly divided into modeling group(n=50) and sham operation group(n=10). The sepsis rat model was constructed by cecal ligation and perforation in the modeling group. The successfully modeled rats were randomly divided into WYZSG low-, medium-and high-dose groups, model group and positive control group. Rats in the sham operation group underwent opening and cecum division but without perforation and ligation. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of rat myocardial tissue. Myocardial cell apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL) assay. Real-time quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression of miR-132-3p and the mRNA expressions of UCP2, microtubule-associated protein light chain 3(LC3-Ⅱ/LC3-Ⅰ), Beclin-1 and caspase-3 in rat myocardial tissue. The protein expressions of UCP2, LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 in myocardial tissue were detected by Western blot. Dual luciferase reporter assay was used to verify the regulatory relationship between miR-132-3p and UCP2. The myocardial fibers of sepsis model rats were disordered, and there were obvious inflammatory cell infiltration as well as myocardial cell edema and necrosis. With the increase of the WYZSG dose, the histopathological changes of myocardium were improved to varying degrees. Compared with the conditions in the sham operation group, the survival rate and left ventricular ejection fraction(LVEF) of rats in the model group, positive control group and WYZSG low-, medium-and high-dose groups were decreased, and the myocardial injury score and apoptosis rate were increased. Compared with the model group, the positive control group and WYZSG low-, medium-and high-dose groups had elevated survival rate and LVEF, and lowered myocardial injury score and apoptosis rate. The expression of miR-132-3p and the mRNA and protein expressions of UCP2 in myocardial tissue in the model group, positive control group and WYZSG low-, medium-and high-dose groups were lower, while the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 were higher than those in the sham operation group. Compared with model group, the positive control group and the WYZSG low-, medium-and high-dose groups had an up-regulation in the expression of miR-132-3p and the mRNA and protein expressions of UCP2, while a down-regulation in the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3. WYZSG inhibited excessive autophagy and apoptosis of myocardial cells in septic rats and improved myocardial injury, possibly by regulating the expression of miR-132-3p/UCP2.

Tanshinone IIA inhibits hypoxia/reoxygenation-induced cardiomyocyte apoptosis and autophagy by regulating ABCE1

To investigate the effects of tanshinone IIA on apoptosis and autophagy induced by hypoxia/reoxygenation in H9C2 cardiomyocytes and its mechanism. H9C2 cardiomyocytes in logarithmic growth phase were divided into control group, hypoxia/reoxygenation model group and tanshinone IIA low-dose, medium-dose and high-dose groups (50, 100, 200 mg/L tanshinone IIA were treated after hypoxia/reoxygenation respectively). The dose with good therapeutic effect was selected for follow-up study. The cells were divided into control group, hypoxia/reoxygenation model group, tanshinone IIA+pcDNA3.1-NC group and tanshinone IIA+pcDNA3.1-ABCE1 group. The cells were transfected with the overexpressed plasmids pcDNA3.1-ABCE1 and pcDNA3.1-NC and then treated accordingly. Cell counting kit-8 (CCK-8) was used to detect H9C2 cell activity in each group. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The ATP-binding cassette transporter E1 (ABCE1), apoptosis-related proteins Bcl-2 and Bax, caspase-3, autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3II/I) and p62 mRNA expression level of H9C2 cells in each group were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expression levels of the above indexes in H9C2 cells were detected by Western blotting. (1) Cell activity and ABCE1 expression: tanshinone IIA inhibited the activity of H9C2 cells induced by hypoxia/reoxygenation, and the effect was significant at medium-dose [(0.95±0.05)% vs. (0.37±0.10)%, P < 0.01], mRNA and protein expression of ABCE1 were significantly reduced [ABCE1 mRNA (2-ΔΔCt): 2.02±0.13 vs. 3.74±0.17, ABCE1 protein (ABCE1/GAPDH): 0.46±0.04 vs. 0.68±0.07, both P < 0.05]. (2) Expression of apoptosis-related proteins: medium-dose of tanshinone IIA inhibited the apoptosis of H9C2 cells induced by hypoxia/reoxygenation [apoptosis rate: (28.26±2.52)% vs. (45.27±3.07)%, P < 0.05]. Compared with the hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated the protein expression of Bax and caspase-3 in H9C2 cells induced by hypoxia/reoxygenation, and significantly up-regulated the protein expression of Bcl-2 [Bax (Bax/GAPDH): 0.28±0.03 vs. 0.47±0.03, caspase-3 (caspase-3/GAPDH): 0.31±0.02 vs. 0.44±0.03, Bcl-2 (Bcl-2/GAPDH): 0.53±0.02 vs. 0.37±0.05, all P < 0.05]. (3) Expression of autophagy-related proteins: compared with the control group, the positive rate of LC3 in the hypoxia/reoxygenation model group was significantly increased, while the positive rate of LC3 in the medium-dose of tanshinone IIA group was significantly decreased [(20.67±3.09)% vs. (42.67±3.86)%, P < 0.01]. Compared with hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated Beclin-1, LC3II/I and p62 protein expressions [Beclin-1 (Beclin-1/GAPDH): 0.27±0.05 vs. 0.47±0.03, LC3II/I ratio: 0.24±0.05 vs. 0.47±0.04, p62 (p62/GAPDH): 0.21±0.03 vs. 0.48±0.02, all P < 0.05]. (4) Expression of apoptosis and autophagy related proteins after transfection with overexpressed ABCE1 plasmid: compared with tanshinone IIA+pcDNA3.1-NC group, the protein expression levels of Bax, caspase-3, Beclin-1, LC3II/I and p62 in tanshinone IIA+pcDNA3.1-ABCE1 group were significantly up-regulated, while the protein expression level of Bcl-2 was significantly down-regulated. 100 mg/L tanshinone IIA could inhibit autophagy and apoptosis of cardiomyocytes by regulating the expression level of ABCE1. So, it protects H9C2 cardiomyocytes injury induced by hypoxia/reoxygenation.

The acute oral toxicity test of ethanol extract of salt-processed Psoraleae Fructus and its acute hepatotoxicity and nephrotoxicity risk assessment.

Psoraleae Fructus is a well-known Traditional Chinese Medicine which has long been used to warm and tonify the kidney and treat diseases such as osteoporosis and diarrhea. However, it may cause multiorgan injury, which limited its use. The aim of this study was to identify the components of ethanol extract of salt-processed Psoraleae Fructus (EEPF) and systematically investigate its acute oral toxicity and the mechanism underlying its acute hepatotoxicity. In this study, the UHPLC-HRMS analysis was carried out for components identification. Followed by acute oral toxicity test in Kunming mice, which received oral gavage of EEPF from 3.85 to 78.00g/kg. Body weight, organ indexes, biochemical analysis, morphology, histopathology, oxidative stress state, TUNEL, mRNA and protein expression of NLRP3/ASC/Caspase-1/GSDMD signaling pathway were evaluated to study the EEPF-induced acute hepatotoxicity and its underlying mechanisms. The results showed that 107 compounds such as psoralen and isopsoralen were identified in EEPF. And the acute oral toxicity test demonstrated the LD50 of EEPF was 15.95g/kg in Kunming mice. The survival mice displayed non-significant difference in body weight compared with Control at the end of the observation period. And the organ indexes of heart, liver, spleen, lung, and kidney showed no significant difference. However, the morphological and histopathological changes of these organs in high-dose-groups mice indicated that the liver and kidney might be the main target toxic organs of EEPF, which showed hepatocyte degeneration with lipid droplets and protein cast in kidney. It could be confirmed by the significant increases of liver and kidney function parameters such as AST, ALT, LDH, BUN, and Crea. In addition, the oxidative stress markers, MDA in the liver and kidney was significantly increased while SOD, CAT, GSH-Px (only liver), and GSH were significantly decreased. Furthermore, EEPF increased the TUNEL-positive cells and the mRNA and protein expression of NLRP3, Caspase-1, ASC and GSDMD in liver with increased protein expression of IL-1β and IL-18. Notably, cell viability test showed that the specific inhibitor of Caspase-1 could reverse the Hep-G2 cell death induced by EEPF. To summarize, this study analyzed the 107 compounds of EEPF. The acute oral toxicity test demonstrated the LD50 value of EEPF was 15.95g/kg in Kunming mice and the liver and kidney might be the main target toxic organs of EEPF. It caused liver injury through oxidative stress and pyroptotic damage via NLRP3/ASC/Caspase-1/GSDMD signaling pathway.

Astragaloside IV ameliorate acute alcohol-induced liver injury in mice via modulating gut microbiota and regulating NLRP3/caspase-1 signaling pathway

Purpose Astragaloside IV (AS-IV) is a natural saponin substance extracted from the plant Radix Astragali with anti-inflammatory, antioxidant, anti-apoptotic, and liver-protecting effects. This study was to evaluate the liver protection effect of AS-IV on mice after acute alcohol stimulation. Materials and Methods Mice were orally administrated with AS-IV (50, 150, and 500 mg/kg, respectively), and sodium carboxymethyl cellulose (CMC, 50 mg/kg) daily for 7 days, before giving five alcohol-intragastric injections. Results Results suggested that the levels of serum ALT and AST, liver SOD, GSH-PX, 4-HNE, and MDA, serum and liver TNF-α, IL-1β, and IL-6, serum lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP), diamine oxidase (DAO) and Myeloperoxidase (MPO), the mRNA and protein expression of hepatic NLRP3, Caspase-1, IL-1β, and IL-18 were significantly decreased in AS-IV-treated mice compared with the model group. Moreover, the effect of AS-IV on histopathology of liver tissue confirmed its protective function. Furthermore, AS-IV ameliorated the gut microbiota imbalance and adjusted the abundance of the following dysfunctional bacteria closer to the control group: Butyricicoccus, Turicibacter, Akkermansia, Anaerotruncus, and Mucispirillum. A strong correlation between intestinal bacteria and potential biomarkers was found. Conclusion Together, our findings indicated that AS-IV exert the hepatoprotective effect by modulating the gut microbiota imbalance and regulating NLRP3/Caspase-1 signaling pathway.