Investigation of Antiproliferative Effects of Combinations of White and Black Garlic Extracts with 5-Fluorouracil (5-FU) on Caco-2 Colorectal Adenocarcinoma Cells.

Garlic is rich in bioactive compounds that are effective against colon cancer cells. This study tests the antioxidant and antiproliferative effects of cold-extracted white and black garlic extracts. Black garlic extracted in water (SSU) exhibits the highest antioxidant activity, phenolic content, and flavonoid content, while black garlic extracted in ethanol (SET) shows the lowest values. Caspase-3 activity is notably higher in the white garlic extracted in methanol (BME), white garlic extracted in methanol combines with 5-FU, black garlic extracted in ethanol (SET), black garlic extracted in ethanol combines with 5-fluorouracil (5-FU), and 5-FU treatments compare to the control group (p > 0.05). BME+5-FU displays the highest caspase-8 activity (p < 0.05). A decrease in NF-κB levels is observed in the SET+5-FU group (p>0.05), while COX-2 activities decrease in the BME, SET+5-FU, SET, and 5-FU groups (p>0.05). Wound healing increases in the BME, BME+5-FU, SET+5-FU, and 5-FU groups (p < 0.05). In conclusion, aqueous black garlic extract may exhibit pro-oxidant activity despite its high antioxidant capacity. It is worth noting that exposure to heat-treated food and increased sugar content may lead to heightened inflammation and adverse health effects. This study is the first to combine garlic with chemo-preventive drugs like 5-FU in Caco-2 cells.

Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Network pharmacology combined with experimental validation to investigate the effect of Rongjin Niantong Fang on chondrocyte apoptosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a chronic degenerative disease that affects the quality of life of middle­aged and elderly individuals, and is one of the major factors leading to disability. Rongjin Niantong Fang (RJNTF) can alleviate the clinical symptoms of patients with KOA, but the molecular mechanism underlying its beneficial effects on KOA remains unknown. Using pharmacological analysis and in vitro experiments, the active components of RJNTF were analyzed to explore their potential therapeutic targets and mechanisms in KOA. The potential targets and core signaling pathways by which RJNTF exerts its effects on KOA were obtained from databases such as Gene Expression Omnibus, Traditional Chinese Medicine Systems Pharmacology and Analysis Platform. Subsequently, chondrocyte apoptosis was modeled using hydrogen peroxide (H2O2). Cell Counting Kit­8 assay involving a poly [ADP­ribose] polymerase­1 (PARP1) inhibitor, DAPI staining, reverse transcription­quantitative PCR, Annexin V­FITC/PI staining and flow cytometry, western blotting and co­immunoprecipitation analysis were used to determine the therapeutic efficacy of RJNTF on KOA and to uncover the molecular mechanism. It was found that PARP1­knockdown lentivirus, incubation with PARP1 inhibitor PJ34, medium and high doses of RJNTF significantly reduced H2O2­induced chondrocyte apoptosis. Medium and high doses of RJNTF downregulated the expression of cleaved caspase­3, cleaved PARP1 and PAR total proteins, as well as nucleus proteins of apoptosis­inducing factor (AIF) and migration inhibitory factor (MIF), and upregulated the expression of caspase­3, PARP1 total protein, as well as the cytoplasmic expression of AIF and MIF, suggesting that RJNTF may inhibit chondrocyte apoptosis through the PARP1/AIF signaling pathway.

[Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells].

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

[Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis].

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

[Chaihu Sanshen Capsules protect rats from myocardial ischemia reperfusion injury via PKCßâ ¡/NOX2/ROS signaling pathway].

This study aims to explore the pathogenesis of myocardial ischaemia reperfusion injury(MIRI) based on oxidative stress-mediated programmed cell death and the mechanism and targets of Chaihu Sanshen Capsules in treating MIRI via the protein kinase Cß(PKCßⅡ)/NADPH oxidase 2(NOX2)/reactive oxygen species(ROS) signaling pathway. The rat model of MIRI was established by the ligation of the left anterior descending branch. Rats were randomized into 6 groups: sham group, model group, clinically equivalent-, high-dose Chaihu Sanshen Capsules groups, N-acetylcysteine group, and CGP53353 group. After drug administration for 7 consecutive days, the area of myocardial infarction in each group was measured. The pathological morphology of the myocardial tissue was observed by hematoxylin-eosin(HE) staining. The apoptosis in the myocardial tissue was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). Enzyme-linked immunosorbent assay(ELISA) was employed to measure the le-vels of indicators of myocardial injury and oxidative stress. The level of ROS was detected by flow cytometry. The protein and mRNA levels of the related proteins in the myocardial tissue were determined by Western blot and real-time quantitative PCR(RT-qPCR), respectively. Compared with the sham group, the model group showed obvious myocardial infarction, myocardial structural disorders, interstitial edema and hemorrhage, presence of a large number of vacuoles, elevated levels of myocardial injury markers, myocardial apoptosis, ROS, and malondialdehyde(MDA), lowered superoxide dismutase(SOD) level, and up-regulated protein and mRNA le-vels of PKCßⅡ, NOX2, cysteinyl aspartate specific proteinase-3(caspase-3), and acyl-CoA synthetase long-chain family member 4(ACSL4) in the myocardial tissue. Compared with the model group, Chaihu Sanshen Capsules reduced the area of myocardial infarction, alleviated the pathological changes in the myocardial tissue, lowered the levels of myocardial injury and oxidative stress indicators and apoptosis, and down-regulated the mRNA and protein levels of PKCßⅡ, NOX2, caspase-3, and ACSL4 in the myocardial tissue. Chaihu Sanshen Capsules can inhibit oxidative stress and programmed cell death(apoptosis, ferroptosis) by regulating the PKCßⅡ/NOX2/ROS signaling pathway, thus mitigating myocardial ischemia reperfusion injury.

Hyperthermia and cisplatin combination therapy promotes caspase-8 accumulation and activation to enhance apoptosis and pyroptosis in cancer cells.

BACKGROUND: Hyperthermia can play a synergistic role with chemotherapy in combination therapy. Although the association between caspase activation, apoptosis, and pyroptosis have been published for both cisplatin (CDDP) and hyperthermia therapies independently, the interactions between these molecular pathways in combination therapy are unknown. The present study aimed to investigate the possible interactions between caspase 8 activation, apoptosis, and pyroptosis in combination therapy. METHODS: Cells were treated with CDDP (15 µg/ml), followed by hyperthermia at optimized temperature (42.5 °C) in water-bath. After combination therapy, cell viability was analyzed by CCK-8, and cell death was analyzed by Annexin-V-FITC/PI and caspases activation. Immuno-staining and co-immuno-precipitation were used to examine the interaction between p62 and caspase-8. Pyroptosis was investigated by western blotting and transmission electron microscopy. E3 ligase Cullin 3 was knockdown by siRNA. In addition, caspase-8 activation was modulated by CRISPR-Cas9 gene-editing or pharmacological inhibition. RESULTS: Combination therapy promoted K63-linked polyubiquitination of caspase-8 and cellular accumulation of caspase-8. In turn, polyubiquitinated caspase-8 interacted with p62 and led to the activation of caspase-3. Knockdown of the E3 ligase Cullin 3 by siRNA reduced caspase-8 polyubiquitination and activation. In addition, combination therapy induced release of the pore-forming N-terminus from gasdermins and promoted pyroptosis along with caspase-8 accumulation and activation. Knockdown of caspase-8 by CRISPR/Cas9 based gene editing reduced the sensitivity of tumor cells to apoptosis and pyroptosis. CONCLUSIONS: Our study presented a novel mechanism in which hyperthermia synergized with chemotherapy in promoting apoptosis and pyroptosis in a caspase-8 dependent manner.

S100A9 exacerbates sepsis-induced acute lung injury via the IL17-NFκB-caspase-3 signaling pathway.

BACKGROUND: Sepsis-induced acute lung injury (ALI) is associated with considerable morbidity and mortality in critically ill patients. S100A9, a key endothelial injury factor, is markedly upregulated in sepsis-induced ALI; however, its specific mechanism of action has not been fully elucidated. METHODS: The Gene Expression Omnibus database transcriptome data for sepsis-induced ALI were used to screen for key differentially expressed genes (DEGs). Using bioinformatics analysis methods such as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction network analyses, the pathogenesis of sepsis-induced ALI was revealed. Intratracheal infusion of lipopolysaccharide (LPS, 10 mg/kg) induced ALI in wild-type (WT) and S100A9 knockout mice. Multiomics analyses (transcriptomics and proteomics) were performed to investigate the potential mechanisms by which S100A9 exacerbates acute lung damage. Hematoxylin-eosin, Giemsa, and TUNEL staining were used to evaluate lung injury and cell apoptosis. LPS (10 µg/mL)-induced murine lung epithelial MLE-12 cells were utilized to mimic ALI and were modulated by S100A9 lentiviral transfection. The impact of S100A9 on cell apoptosis and inflammatory responses were identified using flow cytometry and PCR. The expression of interleukin (IL)-17-nuclear factor kappa B (NFκB)-caspase-3 signaling components was identified using western blotting. RESULTS: Six common DEGs (S100A9, S100A8, IFITM6, SAA3, CD177, and MMP9) were identified in the six datasets related to ALI in sepsis. Compared to WT sepsis mice, S100A9 knockout significantly alleviated LPS-induced ALI in mice, with reduced lung structural damage and inflammatory exudation, decreased exfoliated cell and protein content in the lung lavage fluid, and reduced apoptosis and necrosis of pulmonary epithelial cells. Transcriptomic analysis revealed that knocking out S100A9 significantly affected 123 DEGs, which were enriched in immune responses, defense responses against bacteria or lipopolysaccharides, cytokine-cytokine receptor interactions, and the IL-17 signaling pathway. Proteomic analysis revealed that S100A9 knockout alleviated muscle contraction dysfunction and structural remodeling in sepsis-induced ALI. Multiomics analysis revealed that S100A9 may be closely related to interferon-induced proteins with tetratricopeptide repeats and oligoadenylate synthase-like proteins. LPS decreased MLE12 cell activity, accompanied by high expression of S100A9. The expression of IL-17RA, pNFκB, and cleaved-caspase-3 were increased by S100A9 overexpression and reduced by S100A9 knockdown in LPS-stimulated MLE12 cells. S100A9 knockdown decreases transcription of apoptosis-related markers Bax, Bcl and caspase-3, alleviating LPS-induced apoptosis. CONCLUSIONS: S100A9 as a key biomarker of sepsis-induced acute lung injury, and exacerbates lung damage and epithelial cell apoptosis induced by LPS via the IL-17-NFκB-caspase-3 signaling pathway.

Erythropoietin alleviates lung ischemia-reperfusion injury by activating the FGF23/FGFR4/ERK signaling pathway.

Background: The purpose of the present study was to investigate the effect of erythropoietin (EPO) on lung ischemia-reperfusion injury (LIRI). Methods: Sprague Dawley rats and BEAS-2B cells were employed to construct an ischemia-reperfusion (I/R)-induced model in vivo and in vitro, respectively. Afterward, I/R rats and tert-butyl hydroperoxide (TBHP)-induced cells were treated with different concentrations of EPO. Furthermore, 40 patients with LIRI and healthy controls were enrolled in the study. Results: It was observed that lung tissue damage, cell apoptosis and the expression of BAX and caspase-3 were higher in the LIRI model in vivo and in vitro than in the control group, nevertheless, the Bcl-2, FGF23 and FGFR4 expression level was lower than in the control group. EPO administration significantly reduced lung tissue damage and cell apoptosis while also up-regulating the expression of FGF23 and FGFR4. Rescue experiments indicated that EPO exerted a protective role associated with the FGF23/FGFR4/p-ERK1/2 signal pathway. Notably, the expression of serum EPO, FGF23, FGFR4 and Bcl-2 was decreased in patients with LIRI, while the expression of caspase-3 and BAX was higher. Conclusion: EPO could effectively improve LIRI, which might be related to the activation of the FGF23/FGFR4/p-ERK1/2 signaling pathway.

Reduced expressions of apoptosis-related proteins TRAIL, Bcl-2, and TNFR1 in NK cells of juvenile-onset systemic lupus erythematosus patients: relations with disease activity, nephritis, and neuropsychiatric involvement.

Background: Lupus pathogenesis is mainly ascribed to increased production and/or impaired clearance of dead cell debris. Although self-reactive T and B lymphocytes are critically linked to lupus development, neutrophils, monocytes, and natural killer (NK) cells have also been implicated. This study assessed apoptosis-related protein expressions in NK cells of patients with juvenile-onset systemic lupus erythematosus (jSLE) and relations to disease activity parameters, nephritis, and neuropsychiatric involvement. Methods: Thirty-six patients with jSLE, 13 juvenile dermatomyositis (JDM) inflammatory controls, and nine healthy controls had Fas, FasL, TRAIL, TNFR1, Bcl-2, Bax, Bim, and caspase-3 expressions in NK cells (CD3-CD16+CD56+) simultaneously determined by flow cytometry. Disease activity parameters included Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score, erythrocyte sedimentation rate, C-reactive protein level, anti-double strain DNA antibody level, complement fractions C3 and C4 levels. Results: Patients with jSLE had a profile of significantly reduced expression of TRAIL, Bcl-2, and TNFR1 proteins in NK cells when compared to healthy controls. Similar profile was observed in patients with jSLE with active disease, positive anti-dsDNA, nephritis, and without neuropsychiatric involvement. Patients with jSLE with positive anti-dsDNA also had reduced expression of Bax in NK cells when compared healthy controls and to those with negative anti-dsDNA. Yet, patients with jSLE with negative anti-dsDNA had reduced mean fluorescence intensity (MFI) of Bim in NK cells compared to healthy controls. Patients with jSLE with nephritis also had reduced MFI of Fas in NK cells when compared to those without nephritis. In addition, in patients with jSLE, the proportion of FasL-expressing NK cells directly correlated with the SLEDAI-2K score (rs = 0.6, p = 0.002) and inversely correlated with the C3 levels (rs = -0.5, p = 0.007). Moreover, patients with jSLE had increased NK cell percentage and caspase-3 protein expression in NK cells when compared to JDM controls. Conclusion: This study extends to NK cells an altered profile of TRAIL, Bcl-2, TNFR1, Fas, FasL, Bax, Bim, and caspase-3 proteins in patients with jSLE, particularly in those with active disease, positive anti-dsDNA, nephritis, and without neuropsychiatric involvement. This change in apoptosis-related protein expressions may contribute to the defective functions of NK cells and, consequently, to lupus development. The full clarification of the role of NK cells in jSLE pathogenesis may pave the way for new therapies like those of NK cell-based.

Peste des petits ruminants virus infection induces endoplasmic reticulum stress and apoptosis via IRE1-XBP1 and IRE1-JNK signaling pathways.

BACKGROUND: Peste des petits ruminants (PPR) is a contagious and fatal disease of sheep and goats. PPR virus (PPRV) infection induces endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The activation of UPR signaling pathways and their impact on apoptosis and virus replication remains controversial. OBJECTIVES: To investigate the role of PPRV-induced ER stress and the IRE1-XBP1 and IRE1-JNK pathways and their impact on apoptosis and virus replication. METHODS: The cell viability and virus replication were assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, immunofluorescence assay, and Western blot. The expression of ER stress biomarker GRP78, IRE1, and its downstream molecules, PPRV-N protein, and apoptosis-related proteins was detected by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. 4-Phenylbutyric acid (4-PBA) and STF-083010 were respectively used to inhibit ER stress and IRE1 signaling pathway. RESULTS: The expression of GRP78, IRE1α, p-IRE1α, XBP1s, JNK, p-JNK, caspase-3, caspase-9, Bax and PPRV-N were significantly up-regulated in PPRV-infected cells, the expression of Bcl-2 was significantly down-regulated. Due to 4-PBA treatment, the expression of GRP78, p-IRE1α, XBP1s, p-JNK, caspase-3, caspase-9, Bax, and PPRV-N were significantly down-regulated, the expression of Bcl-2 was significantly up-regulated. Moreover, in PPRV-infected cells, the expression of p-IRE1α, p-JNK, Bax, and PPRV-N was significantly decreased, and the expression of Bcl-2 was increased in the presence of STF-083010. CONCLUSIONS: PPRV infection induces ER stress and IRE1 activation, resulting in apoptosis and enhancement of virus replication through IRE1-XBP1s and IRE1-JNK pathways.

[Reversal Effect of Arctigenin on the Drug Resistance in Leukemia K562/A02 Cells and Its Mechanism].

OBJECTIVE: To study the effect of arctigenin(ARG) on adriamycin(ADM) resistance of leukemia cell line K562/A02 and the underlying mechanism. METHODS: Human leukemia cell line K562 and ADM-resistant cell line K562/A02 were cultured and treated with 2.5-50 µmol/L ADM. Cell proliferation was measured using CCK-8 method, and half maximal inhibitory concentration (IC50) was calculated. K562/A02 cells were treated with different concentrations of ARG (1, 2, 4, 8, 16 mmol/L) to detect the effect of ARG on K562/A02 cells, and a suitable concentration (2 mmol/L) was selected for subsequent experiments. K562/A02 cells were treated with 2 mmol/L ARG and 5 µmol/L ADM, and cell apoptosis was detected by flow cytometry, the expression of P-gp, MRP, cleaved caspase-3, Bax, Bcl-2 proteins and the TLR4/NF-κB signaling pathway-related proteins were measured by Western blot. TLR4 overexpression plasmid was transfected into K562/A02 cells which were co-treated with ARG and ADM, then drug sensitivity and cell apoptosis were measured. RESULTS: The IC50 value of ADM on K562/A02 cells was 36.57 µmol/L, which was significantly higher than that on K562 cells (1.30 µmol/L). ARG with a concentration of ≤2 mmol/L did not have a significant effect on K562/A02 cells. 2 mmol/L ARG significantly reduced the IC50 of ADM on K562/A02 cells. In 5 µmol/L ADM-treated K562/A02 cells, compared with the control group, the apoptosis rate of K562/A02 cells in the ARG group was significantly increased, the expressions of cleaved caspase-3, Bax proteins were significantly upregulated, the expressions of P-gp, MRP, Bcl-2, TLR4, MyD88, and p-NF-κB proteins were significantly downregulated, and the differences were statistically significant (P < 0.05). After transfection with TLR4 overexpression plasmid, the sensitivity of ARG-treated K562/A02 cells to ADM was reduced (P < 0.05), the cell apoptosis was decreased, and the expressions of P-gp, MRP, Bcl-2 and TLR4/NF-κB signaling pathway-related proteins were significantly elevated, while the expressions of cleaved caspase-3 and Bax proteins were significantly decreased (all P < 0.05). CONCLUSION: ARG may reverse the resistance of human leukemia cell line K562/A02 to ADM by inhibiting TLR4/NF-κB signaling pathway.

[Effect of Shikonin on Autophagy and Apoptosis of Human Promyelocytic Leukemia Cells].

OBJECTIVE: To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism. METHODS: Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins. RESULTS: Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05). CONCLUSION: Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Methylmercury Induces Apoptosis in Mouse C17.2 Neural Stem Cells through the Induction of OSGIN1 Expression by NRF2.

Methylmercury is a known environmental pollutant that exhibits severe neurotoxic effects. However, the mechanism by which methylmercury causes neurotoxicity remains unclear. To date, we have found that oxidative stress-induced growth inhibitor 1 (OSGIN1), which is induced by oxidative stress and DNA damage, is also induced by methylmercury. Therefore, in this study, we investigated the relationship between methylmercury toxicity and the induction of OSGIN1 expression using C17.2 cells, which are mouse brain neural stem cells. Methylmercury increased both OSGIN1 mRNA and protein levels in a time- and concentration-dependent manner. Moreover, these increases were almost entirely canceled out by pretreatment with actinomycin D, a transcription inhibitor. Furthermore, similar results were obtained from cells in which expression of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) was suppressed, indicating that methylmercury induces OSGIN1 expression via NRF2. Methylmercury causes neuronal cell death by inducing apoptosis. Therefore, we next investigated the role of OSGIN1 in methylmercury-induced neuronal cell death using the activation of caspase-3, which is involved in apoptosis induction, as an indicator. As a result, the increase in cleaved caspase-3 (activated form) induced by methylmercury exposure was decreased by suppressing OSGIN1, and the overexpression of OSGIN1 further promoted the increase in cleaved caspase-3 caused by methylmercury. These results suggest, for the first time, that OSGIN1 is a novel factor involved in methylmercury toxicity, and methylmercury induces apoptosis in C17.2 cells through the induction of OSGIN1 expression by NRF2.

Integrated analyses for identification of a three-gene signature associated with Chaihu Shugan San formula for hepatocellular carcinoma treatment.

Chaihu Shugan San (CSS) is a well-known traditional herbal formula that has the potential to ameliorate hepatocellular carcinoma (HCC); however, its mechanism of action remains unknown. Here, we identified the key targets of CSS against HCC and developed a prognostic model to predict the survival of patients with HCC. The effect of CSS plus sorafenib on HCC cell proliferation was evaluated using the MTT assay. LASSO-Cox regression was used to establish a three-gene signature model targeting CSS. Correlations between immune cells, immune checkpoints and risk score were determined to evaluate the immune-related effects of CSS. The interactions between the components and targets were validated using molecular docking and Surface Plasmon Resonance (SPR) assays. CSS and sorafenib synergistically inhibited HCC cell proliferation. Ten core compounds and 224 targets were identified using a drug compound-target network. The prognostic model of the three CSS targets (AKT1, MAPK3 and CASP3) showed predictive ability. Risk scores positively correlated with cancer-promoting immune cells and high expression of immune checkpoint proteins. Molecular docking and SPR analyses confirmed the strong binding affinities of the active components and the target genes. Western blot analysis confirmed the synergistic effect of CSS and sorafenib in inhibiting the expression of these three targets. In conclusion, CSS may regulate the activity of immune-related factors in the tumour microenvironment, reverse immune escape, enhance immune responses through AKT1, MAPK3, and CASP3, and synergistically alleviate HCC. The co-administration of sorafenib with CSS has a strong clinical outlook against HCC.

MiR-98-5p plays suppressive effects on IL-1ß-induced chondrocyte injury associated with osteoarthritis by targeting CASP3.

BACKGROUND: This study aims to explore how miR-98-5p affects osteoarthritis, focusing on its role in chondrocyte inflammation, apoptosis, and extracellular matrix (ECM) degradation. METHODS: Quantitative real-time PCR was used to measure miR-98-5p and CASP3 mRNA levels in OA cartilage tissues and IL-1ß-treated CHON-001 cells. We predicted miR-98-5p and CASP3 binding sites using TargetScan and confirmed them via luciferase reporter assays. Chondrocyte viability was analyzed using CCK-8 assays, while pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) were quantified via ELISA. Caspase-3 activity was examined to assess apoptosis, and Western blotting was conducted for protein marker quantification. RESULTS: Our results showed lower miR-98-5p levels in both OA cartilage and IL-1ß-stimulated cells. Increasing miR-98-5p resulted in reduced pro-inflammatory cytokines, decreased caspase-3 activity, and improved cell viability. Furthermore, miR-98-5p overexpression hindered IL-1ß-induced ECM degradation, evident from the decline in MMP-13 and ß-catenin levels, and an increase in COL2A1 expression. MiR-98-5p's impact on CASP3 mRNA directly influenced its expression. Mimicking miR-98-5p's effects, CASP3 knockdown also inhibited IL-1ß-induced inflammation, apoptosis, and ECM degradation. In contrast, CASP3 overexpression negated the suppressive effects of miR-98-5p. CONCLUSIONS: In conclusion, our data collectively suggest that miR-98-5p plays a protective role against IL-1ß-induced damage in chondrocytes by targeting CASP3, highlighting its potential as a therapeutic target for OA.

Circumlimbal suture and laser burns: Comparison between two different chronic glaucoma models. / çŽ¯è§’è†œç¼˜ç¼åˆä¸Žæ¿€å ‰å ‰å‡æ¨¡åž‹ï¼šä¸¤ç§æ ¢æ€§é«˜çœ¼åŽ‹æ¨¡åž‹çš„æ¯”è¾ƒï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Glaucoma is a multifactorial optic neuropathy with a high rate of irreversible visual loss, and its pathogenesis is complex and still unclear. Elevated intraocular pressure (IOP) is well recognized as the sole modifiable risk factor for the development of glaucoma in the majority of cases. This study aims to compare 2 different methods of inducing chronic ocular hypertension by circumlimbal suture or by laser burns in degree and lasting time of the IOP, different status of the retina and retinal ganglion cells (RGCs), and changes of the microstructure of neurons. METHODS: The chronic ocular hypertension models were induced by 2 different ways. One kind of the models was built by unilateral circumlimbal suture (10/0) implantation (suture group), another kind of model was built by laser burns at trabecular meshwork and episcleral veins (laser group). The untreated contralateral eye served as the control group. Changes in IOP were observed and regularly monitored in the 2 groups of rats. HE staining was applied to observe the retinal and optic nerve pathology. Transmission electron microscope (TEM) was used to observe the mitochondrial morphology. RGCs were specifically labeled with Brn3b antibody and counted. The expression of caspase-3 was detected by Western blotting to clarify the apoptosis of RGCs. RESULTS: Compared with the control group, IOP were significantly increased in the suture group and the laser group (both P<0.05). The suture group induced a 1.5-fold elevation of IOP, and sustained for 8 weeks. The laser group induced a 2-fold elevation of IOP for 12 weeks. Both methods could cause RGCs loss (both P<0.05), which were verified by pathology and immune staining of Brn3b. The expressions of caspase-3 were also increased (both P<0.05). The mitochondrial morphology became more fragment, which changed from long shape to round and small one under TEM in 2 models. For comparison, the pathology changes of retinal structure in suture group were not obviously than those in the laser group. CONCLUSIONS: Circumlimbal suture can build an effective model of chronic elevated IOP and induce glaucomatous pathologic changes similar to those in the laser photocoagulation, but the pathologic changes are milder than those in laser photocoagulation. Compare with translimbal laser photocoagulation, equipment and skill demand for circumlimbal suture is less.