OBJECTIVE: To investigate the therapeutic potential of pseudolaric acid B (PAB) on non-alcoholic fatty liver disease (NAFLD) and its underlying molecular mechanism in vitro and in vivo. METHODS: Eight-week-old male C57BL/6J mice (n=32) were fed either a normal chow diet (NCD) or a high-fat diet (HFD) for 8 weeks. The HFD mice were divided into 3 groups according to a simple random method, including HFD, PAB low-dose [10 mg/(kg·d), PAB-L], and PAB high-dose [20 mg/(kg·d), PAB-H] groups. After 8 weeks of treatment, glucose metabolism and insulin resistance were assessed by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Biochemical assays were used to measure the serum and cellular levels of total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). White adipose tissue (WAT), brown adipose tissue (BAT) and liver tissue were subjected to hematoxylin and eosin (H&E) staining or Oil Red O staining to observe the alterations in adipose tissue and liver injury. PharmMapper and DisGeNet were used to predict the NAFLD-related PAB targets. Peroxisome proliferator-activated receptor alpha (PPARα) pathway involvement was suggested by Kyoto Encyclopedia of Genes and Genomes (KEGG) and search tool Retrieval of Interacting Genes (STRING) analyses. Luciferase reporter assay, cellular thermal shift assay (CETSA), and drug affinity responsive target stability assay (DARTS) were conducted to confirm direct binding of PAB with PPARα. Molecular dynamics simulations were applied to further validate target engagement. RT-qPCR and Western blot were performed to assess the downstream genes and proteins expression, and validated by PPARα inhibitor MK886. RESULTS: PAB significantly reduced serum TC, TG, LDL-C, AST, and ALT levels, and increased HDL-C level in HFD mice (P<0.01). Target prediction analysis indicated a significant correlation between PAB and PPARα pathway. PAB direct target binding with PPARα was confirmed through luciferase reporter assay, CETSA, and DARTS (P<0.05 or P<0.01). The target engagement between PAB and PPARα protein was further confirmed by molecular dynamics simulations and the top 3 amino acid residues, LEU321, MET355, and PHE273 showed the most significant changes in mutational energy. Subsequently, PAB upregulated the genes expressions involved in lipid metabolism and mitochondrial biogenesis downstream of PPARα (P<0.05 or P<0.01). Significantly, the PPARα inhibitor MK886 effectively reversed the lipid-lowering and PPARα activation properties of PAB (P<0.05 or P<0.01). CONCLUSION PAB mitigates lipid accumulation, ameliorates liver damage, and improves mitochondrial biogenesis by binding with PPARα, thus presenting a potential candidate for pharmaceutical development in the treatment of NAFLD.
Animals ; PPAR alpha/metabolism* ; Non-alcoholic Fatty Liver Disease/pathology* ; Male ; Mice, Inbred C57BL ; Lipid Metabolism/drug effects* ; Diterpenes/therapeutic use* ; Organelle Biogenesis ; Diet, High-Fat ; Humans ; Mice ; Liver/metabolism* ; Insulin Resistance ; Mitochondria/metabolism* ; Molecular Docking Simulation

OBJECTIVES: To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. METHODS: Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. RESULTS: MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. CONCLUSIONS YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.
Animals ; MicroRNAs/genetics* ; Podocytes/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Autophagy/drug effects* ; Rats, Wistar ; Protein Kinases/metabolism* ; Rats ; Forkhead Box Protein O1 ; Mice ; Mitochondria/drug effects* ; Ubiquitin-Protein Ligases/metabolism* ; Glucose ; Diabetic Nephropathies ; Male ; Membrane Proteins/metabolism* ; Intracellular Signaling Peptides and Proteins

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.
Adult ; Aged ; Aged, 80 and over ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; therapeutic use ; Cofilin 1 ; metabolism ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Mitochondria ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; pathology ; Tumor Cells, Cultured

Ageratina adenophora is a noxious plant and it is known to cause acute asthma, diarrhea, depilation, and even death in livestock (Zhu et al., 2007; Wang et al., 2017). A. adenophora grows near roadsides and degraded land worldwide (He et al., 2015b). In the areas where it grows, A. adenophora is an invasive species that inhibits the growth of local plants and causes poisoning in animals that come in contact with it (Nie et al., 2012). In China, these plants can be found in Yunnan, Sichuan, Guizhou, Chongqing, and other southwestern areas (He et al., 2015a) and they have become a dominant species in these local regions. It threatens the native biodiversity and ecosystem in the invaded areas and causes serious economic losses (Wang et al., 2017). It has been reported that A. adenophora can grow in the northeast direction at a speed of 20 km per year in China (Guo et al., 2009). Because of the damage caused by A. adenophora, it ranks among the earliest alien invasive plant species in China (Wang et al., 2017).
Adenosine Triphosphatases/metabolism* ; Ageratina/toxicity* ; Animals ; Biodiversity ; Chemical and Drug Induced Liver Injury/pathology* ; China ; DNA, Mitochondrial/genetics* ; Ecosystem ; Introduced Species ; Liver/drug effects* ; Mice ; Microscopy, Electron, Transmission ; Mitochondria, Liver/pathology* ; Plant Extracts/toxicity*

To investigate the effect of diosgenin (Dgn) on chondrocytes and its relation to JAK2/STAT3 signaling pathway in mice with osteoarthritis (OA).Fifteen male C57BL/6 mice were randomly divided into three groups:control group, OA group and OA+Dgn group. After 4 weeks of treatment, the histopathological changes of cartilage tissue were observed by toluidine blue staining under light microscopy and the ultrastructure of chondrocytes was observed under electron microscopy. The primarily cultured chondrocytes of OA mice were randomly divided into 4 groups:(1) OA group, (2) Dgn group, (3) Dgn+AG490 group, (4) AG490 group. The expression of p-JAK2, p-STAT3, Bax, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) were detected by Western blotting, and superoxide dismutase (SOD) was detected using colorimetric method.The morphological observation showed that the chondrocytes of OA group presented considerable pathological changes, while the chondrocytes in OA+Dgn group maintained intact membrane. Electron microscopy observation found obvious injury in cartilage tissues of OA group, while that in OA+Dgn group remained smooth. Compared with OA group, the expressions of p-JAK2 and p-STAT3 in chondrocytes of Dgn group were increased (all<0.05), and the expressions of Bax protein, SDH, COX and SOD were decreased (all<0.05). While compared with Dgn group, the expressions of p-JAK2, p-STAT3, SDH, COX and SOD in chondrocytes of Dgn+AG490 group were decreased (all<0.05), and the expression of Bax protein was increased (<0.05).Diosgenin can inhibit apoptosis and increase mitochondrial oxidative stress capacity of chondrocytes in mice with osteoarthritis, which is closely related to the activation of JAK2/STAT3 signaling pathway.
Animals ; Apoptosis ; drug effects ; Cartilage ; drug effects ; pathology ; Chondrocytes ; chemistry ; drug effects ; pathology ; Diosgenin ; pharmacology ; Electron Transport Complex IV ; metabolism ; Janus Kinase 2 ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria ; drug effects ; genetics ; Osteoarthritis ; genetics ; physiopathology ; Oxidative Stress ; drug effects ; STAT3 Transcription Factor ; drug effects ; Signal Transduction ; Succinate Dehydrogenase ; metabolism ; Superoxide Dismutase ; metabolism ; Tyrphostins ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc and its possible molecular mechanisms in vitro and in vivo.

METHODSTransonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb. et Zucc, and the content of toosendanin in the crude extract was measured by high performance liquid chromatography (HPLC). Anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc were investigated in in vivo and in vitro studies. In the in vitro experiment, human hepatocellular carcinoma cell lines SMMC-7721 and Hep3B were co-incubated with toosendanin crude extract of different concentrations, respectively. In the in vivo experiment, BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract.

RESULTSHPLC revealed the content of toosendanin was about 15%. Crude extract from Melia toosendan Sieb. et Zucc inhibited cancer cells growth in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50, 72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3B cells. Both high-dose [0.69 mg/(kg d)] and low-dose [0.138 mg/(kg d)] crude extract could markedly suppress cancer growth, and the inhibition rate was greater than 50%. Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies. Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced.

CONCLUSIONSToosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro. The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; ultrastructure ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Female ; Immunohistochemistry ; Liver Neoplasms ; drug therapy ; pathology ; ultrastructure ; Male ; Melia ; chemistry ; Mice, Inbred BALB C ; Mitochondria ; drug effects ; metabolism ; Neoplasm Transplantation ; Plant Extracts ; therapeutic use ; Reference Standards ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the effects of Heijiangdan Ointment ( HJD) on oxidative stress in (60)Co γ-ray radiation-induced dermatitis in mice.

METHODSFemale Wistar mice with grade 4 radiation dermatitis induced by (60)Co γ-rays were randomly divided into four groups (n=12 per group); the HJD-treated, recombinant human epidermal growth factor (rhEGF)-treated, Trolox-treated, and untreated groups, along with a negative control group. On the 11th and 21st days after treatment, 6 mice in each group were chosen for evaluation. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH) were detected using spectrophotometric methods. The fibroblast mitochondria were observed by transmission electron microscopy (TEM). The expressions of fibroblast growth factor 2 (FGF-2) and transforming growth factor β1 (TGF-β1) were analyzed by western blot.

RESULTSCompared with the untreated group, the levels of SOD, MDA and LDH, on the 11th and 21st days after treatment showed significant difference (P<0.05). TEM analysis indicated that fibroblast mitochondria in the untreated group exhibited swelling and the cristae appeared fractured, while in the HJD group, the swelling of mitochondria was limited and the rough endoplasmic reticulum appeared more relaxed. The expressions of FGF-2 and TGF-β1 increased in the untreated group compared with the negative control group (P<0.05). After treatment, the expression of FGF-2, rhEGF and Trolox in the HJD group were significantly increased compared with the untreated group (P<0.05), or compared with the negative control group (P<0.05). The expression of TGF-β1 showed significant difference between untreated and negative control groups (P<0.05). HJD and Trolox increased the level of TGF-β1 and the difference was marked as compared with the untreated and negative control groups (P<0.05).

CONCLUSIONHJD relieves oxidative stress-induced injury, increases the antioxidant activity, mitigates the fibroblast mitochondrial damage, up-regulates the expression of growth factor, and promotes mitochondrial repair in mice.

Animals ; Biological Products ; pharmacology ; therapeutic use ; Cell Proliferation ; drug effects ; radiation effects ; Cobalt Radioisotopes ; Dermatitis ; complications ; drug therapy ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Fibroblasts ; drug effects ; pathology ; radiation effects ; Gamma Rays ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Mice ; Mitochondria ; drug effects ; metabolism ; radiation effects ; Ointments ; Oxidative Stress ; drug effects ; radiation effects ; Pharmaceutical Preparations ; Radiation Injuries ; complications ; drug therapy ; pathology ; Superoxide Dismutase ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Up-Regulation ; drug effects ; radiation effects

OBJECTIVETo investigate the cytoprotective effects of Saeng-kankunbi-tang (, SKT), a herbal prescription consisting of Artemisia capillaris and Alisma canaliculatum, and its underlying mechanism involved.

METHODSIn mice, blood biochemistry and histopathology were assessed in carbon tetrachloride (CCl4)-induced oxidative hepatic injury in vivo. The animal groups included vehicle-treated control, CCl4, SKT 500 mg/(kg day) CCl4+SKT 200 or 500 mg/(kg day). In HepG2 cell, tert-butyl hydroperoxide (tBHP) induced severe oxidative stress and mitochondrial dysfunction in vitro. The cyto-protective effects of SKT were determined by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay, flfluorescence activated cell sorting analysis and western blotting.

RESULTSThe administration of SKT prevented liver damage induced by CCl4 in mice, by inhibition of hepatocyte degeneration and inflflammatory cell infifiltration as well as plasma parameters such as alanine aminotransferase (P<0.01). Moreover, treatment with tBHP induced hepatocyte death and cellular reactive oxygen species production in hepatocyte cell line. However, SKT pretreatment (30-300 μg/mL) reduced this cell death and oxidative stress (P<0.01). More importantly, SKT inhibited the ability of tBHP to induce changes in mitochondrial membrane transition in cell stained with rhodamine 123 P<0.01). Furthermore, treatment with SKT induced extracellular signal-regulated kinases-mediated nuclear factor erythroid-2-related factor 2 (Nrf2) activation as well as the expressions of heme oxygenase 1 and glutamate- cystein ligase catalytic, Nrf2 target genes.

CONCLUSIONSSKT has the ability to protect hepatocyte against oxidative stress and mitochondrial damage mediated by Nrf2 activation.

Animals ; Antioxidants ; pharmacology ; Carbon Tetrachloride ; Cell Death ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hep G2 Cells ; Humans ; Liver ; drug effects ; enzymology ; pathology ; MAP Kinase Signaling System ; drug effects ; Mice, Inbred C57BL ; Mitochondria ; drug effects ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; drug effects ; Peroxides ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; Reactive Oxygen Species ; metabolism

OBJECTIVEHomeopathic nosodes have seldom been scientifically validated for their anticancer effects. This study was conducted to examine if a recently developed hepatitis C nosode has demonstrable anticancer potential in cancer cells in vitro.

METHODSAnticancer effects of Hepatitis C 30C (Hep C 30), if any, were initially tested on three cancer cell lines, HepG2 (liver cancer), MCF-7 (breast cancer) and A549 (lung cancer) and one normal liver cell line WRL-68 cells and subsequently a more thorough study using further scientific protocols was undertaken on HepG2 cells (against WRL-68 cells as the normal control) as HepG2 cells showed better anticancer response than the other two. Three doses, one at 50% lethal dose (LD50) and the other two below LD50, were used on HepG2 cells subsequently. Protocols like apoptosis induction and its possible signaling mechanism were deployed using immunoblots of relevant signal proteins and confocal microscopy, with particular reference to telomerase and topoisomerase II (Top II) activities, two strong cancer biomarkers for their direct relationship with divisional activities of cells and DNAs.

RESULTSHep C 30 induced apoptosis, caused distorted cell morphology typical of apoptotic cells, increased reactive oxygen species generation and produced increased DNA nicks. Further it enhanced pro-apototic signal proteins like Bax, cytochrome c and inhibited anti-apoptotic signal proteins, Bcl-2, cytochrome c and caspase-3, changed mitochondrial membrane potential and caused externalization of phosphatidylserine. The drug also decreased expression of two cancer biomarkers, Top II and telomerase, consistent with its anticancer effect.

CONCLUSIONHep C 30 has demonstrable anticancer effects against liver cancer cells in vitro.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; DNA Topoisomerases, Type II ; metabolism ; Hep G2 Cells ; Hepacivirus ; Humans ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Materia Medica ; Mitochondria ; drug effects ; physiology ; Telomerase ; metabolism