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This study explored the protective effect of atractylenolide Ⅰ(AO-Ⅰ) against acetaminophen(APAP)-induced acute liver injury(ALI) in mice and its underlying mechanism. C57 BL/6 J mice were randomly divided into a control group, an APAP group(500 mg·kg~(-1)), a low-dose combination group(500 mg·kg~(-1) APAP + 60 mg·kg~(-1) AO-Ⅰ), and a high-dose combination group(500 mg·kg~(-1) APAP + 120 mg·kg~(-1) AO-Ⅰ). ALI was induced by intraperitoneal injection of APAP(500 mg·kg~(-1)). AO-Ⅰ by intragastric administration was performed 2 hours before APAP treatment, and the control group received the same dose of solvent by intragastric administration or intraperitoneal injection. The protective effect of AO-Ⅰ against APAP-induced ALI was evaluated by detecting alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels in the plasma and Hamp;E staining in liver tissues of mice. The malondialdehyde(MDA) and glutathione(GSH) content and catalase(CAT) activity in mouse liver tissues were detected to evaluate the effect of AO-Ⅰ on APAP-induced oxidative stress in the liver. The proteins in the liver p38 mitogen-activated protein kinase(p38 MAPK), c-jun N-terminal kinase(JNK), and nuclear factor kappa-B p65(NF-κB p65) signaling pathways were measured by Western blot, and the liver inflammatory cytokines interleukin-1β(IL-1β) and interleukin-6(IL-6) were detected by real-time PCR. Compared with the APAP group, the combination groups showed reduced APAP-induced ALT level and liver MDA content, potentiated liver CAT activity, and elevated GSH content. Mechanistically, AO-Ⅰ treatment significantly inhibited APAP-up-regulated MAPK phosphorylation and NF-κB p65, and significantly reduced the transcriptional activities of IL-1β and IL-6, downstream targets of NF-κB p65. AO-Ⅰ can improve APAP-induced ALI and the underlying mechanism is related to the inhibition of the MAPK/NF-κB p65 signaling pathway in APAP-challenged mice.

Background Pulmonary arterial hypertension (PAH), characterized by pulmonary artery constriction and vascular remodeling, has a high mortality rate. New drugs for the treatment of PAH urgently need to be developed. Purpose This study was designed to investigate the vasorelaxant activity of OTNA in isolated pulmonary arteries, and explore its molecular mechanism. Methods Pulmonary arteries and thoracic aortas were isolated from mice, and vascular tone was tested with a Wire Myograph System. Nitric oxide levels were determined with DAF-FM DA and DAX-J2™ Red. Cellular thermal shift assays, microscale thermophoresis, and molecular docking were used to identify the interaction between OTNA and aryl hydrocarbon receptor (AhR). The levels of PI3K, p-PI3K, Akt, p-Akt, eNOS, p-eNOS, and AhR were analyzed by Western blotting. Results OTNA selectively relaxed the isolated pulmonary artery rings in an endothelium-dependent manner. Mechanistic study showed that OTNA induced NO production through activation of the PI3K/Akt/eNOS pathway in endothelial cells. Furthermore, we also found that OTNA directly bound to AhR and activated the PI3K/Akt/eNOS pathway to dilate pulmonary arteries by inhibiting AhR. Conclusions OTNA relaxes pulmonary arteries by antagonizing AhR. This study provides a new natural antagonist of AhR as a promising lead compound for PAH treatment.

Tanshinone Ⅱ_A( Tan Ⅱ_A),the liposoluble constituents of Salvia miltiorrhiza,can not only ameliorate the lipidic metabolism and decrease the concentration of lipid peroxidation,but also resist oxidation damage,scavenge free radicals and control inflammation,with a protective effect on prognosis after liver function impairment. Therefore,the studies on the exact mechanism of Tan Ⅱ_A in protecting the liver can provide important theoretical and experimental basis for the prevention and treatment effect of Tan Ⅱ_A for liver injury. In the present study,the protective effects and mechanism of Tan Ⅱ_A on 4-hydroxynonenal( 4-HNE)-induced liver injury were investigated in vitro. Normal liver tissues NCTC 1469 cells were used to induce hepatocytes oxidative damages by 4-HNE treatment. The protective effect of Tan Ⅱ_A on hepatocytes oxidative damages was detected by release amount of lactate dehydrogenase( LDH) analysis and hoechst staining. The protein expression changes of peroxisome proliferator-activated receptor α( PPARα) and peroxisome proliferator response element( PPRE) were analyzed by Western blot analysis in NCTC 1469 cells before and after Tan Ⅱ_A treatment. The gene expression changes of fatty aldehyde dehydrogenase( FALDH) were analyzed by Real-time polymerase chain reaction( PCR) analysis. The results showed that 4-HNE increased the release amount of LDH,lowered the cell viability of NCTC 1469 cells,and Tan Ⅱ_A reversed 4-HNE-induced hepatocyte damage. Western blot analysis and RT-PCR analysis results showed that 4-HNE decreased the expression of PPARα and FALDH and increased the expression of 4-HNE. However,the expression of PPARα and FALDH were increased significantly and the expression of 4-HNE was decreased obviously after Tan Ⅱ_A treatment. This study confirmed that the curative effect of Tan Ⅱ_A was obvious on hepatocytes damage,and the mechanism may be associated with activating PPARα and FALDH expression as well as scavenging 4-HNE.

// Yun-fang Zhen 1, * , Song-tao Li 2, * , Yun-rong Zhu 3, * , Xiao-dong Wang 1 , Xiao-zhong Zhou 2 , Lun-qing Zhu 1 1 The Center of Diagnosis and Treatment for Children’s Bone Diseases, The Children’s Hospital Affiliated to Soochow University, Suzhou, China 2 The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, China 3 Department of Orthopedics, The Affiliated Jiangyin Hospital of Medical College of Southeast University, Jiangyin, China * These authors have contributed equally to this work Correspondence to: Lun-qing Zhu, email: zhulunqingszgk@163.com Xiao-dong Wang, email: xiaodongwangsz@163.com Xiao-zhong Zhou, email: zhouxiaozhongorth@163.com Keywords: osteosarcoma (OS), salinomycin, DNA-PKcs, microRNA-101, autophagy Received: September 09, 2016 Accepted: September 26, 2016 Published: October 17, 2016 ABSTRACT Malignant osteosarcoma (OS) is still a deadly disease for many affected patients. The search for the novel anti-OS agent is extremely urgent and important. Our previous study has proposed that salinomycin is a novel anti-OS agent. Here we characterized DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a primary salinomycin resistance factor in OS cells. DNA-PKcs inhibitors (NU7026, NU7441 and LY294002) or DNA-PKcs shRNA knockdown dramatically potentiated salinomycin-induced death and apoptosis of OS cells (U2OS and MG-63 lines). Further, forced-expression of microRNA-101 (“miR-101”) downregulated DNA-PKcs and augmented salinomycin’s cytotoxicity against OS cells. Reversely, over-expression of DNA-PKcs in OS cells inhibited salinomycin’s lethality. For the mechanism study, we show that DNA-PKcs is required for salinomycin-induced pro-survival autophagy activation. DNA-PKcs inhibition (by NU7441), shRNA knockdown or miR-101 expression inhibited salinomycin-induced Beclin-1 expression and autophagy induction. Meanwhile, knockdown of Beclin-1 by shRNA significantly sensitized salinomycin-induced OS cell lethality. In vivo , salinomycin administration suppressed U2OS xenograft tumor growth in severe combined immuno-deficient (SCID) mice, and its anti-tumor activity was dramatically potentiated with co-administration of the DNA-PKcs inhibitor NU7026. Together, these results suggest that DNA-PKcs could be a primary resistance factor of salinomycin in OS cells. DNA-PKcs inhibition or silence may thus significantly increase salinomycin’s sensitivity in OS cells.

This study introduced a prototype of an axial-stress bioreactor system that supports long-term growth and development of engineered tissues. The main features of this bioreactor are an integrated substance exchanger and feedback control of pH and PO₂. A 21-day study was conducted to validate the system's ability to maintain a stable environment, while remaining sterile. Our results showed that the pH, PO₂, and nutrient (glucose) remained balanced at appropriate levels, while metabolic waste (lactic acid) was removed. No bacteria or fungi were detected in the system or tissue; thus, demonstrating that it was sterile. These data indicate the bioreactor's strong potential for long-term tissue culture. To explore this idea, the effect of dynamic culture, including cyclic compression and automatic substance exchange, on mouse bone-marrow mesenchymal stem cells (BMSCs) seeded in decalcified bone matrix was studied using the bioreactor prototype. Histological sections of the engineered tissues showed higher cell densities in scaffolds in dynamic culture compared to those in static culture, while cell cycle analysis showed that dynamic culture promoted BMSC proliferation (proliferation index, PI=34.02±1.77) more effectively than static culture (PI=26.66±1.81). The results from a methyl thiazolyl tetrazolium assay were consistent with the loading experimental data. Furthermore, elevated alkaline phosphatase activity and calcium content were observed in dynamic condition compared to static culture. In conclusion, this bioreactor system supplies a method of modulating the pH and PO₂ in defined ranges with only small fluctuations; it can be used as a physiological or pathological analog. Automatic control of the environment is a practical solution for long-term, steady-state culture for future commercialization.

Dexamethasone (Dex) causes osteoblast cell injuries. In the present research, we tested the potential effect of SC79, a novel and specific Akt activator, against Dex in osteoblasts. In primary murine osteoblasts and osteoblastic MC3T3-E1 cells, pretreatment with SC79 significantly attenuated Dex-induced cell death. Further, Dex-induced mitochondrial permeability transition pore (mPTP) opening, cytochrome C release and apoptosis activation were dramatically alleviated with SC79 pretreatment in above cells. At the molecular level, SC79 activated Akt, which was indispensable for subsequent osteoblast protection against Dex. Akt inhibitors (LY294002, perifosine and MK-2206) blocked SC79-induced Akt activation and abolished its anti-Dex actions in osteoblasts. Further, SC79 activated Akt downstream Nrf2 (NF-E2-related factor 2) signaling and attenuated Dex-induced oxidative stress in osteoblasts. Nrf2 shRNA knockdown or S40T mutation almost reversed SC79-mediated anti-oxidant and cytoprotective activities in osteoblasts. Together, these results suggest that SC79 activates Akt-Nrf2 signaling to protect osteoblasts from Dex.