A novel apoptosis probe, cyclic ApoPep-1, for in vivo imaging with multimodal applications in chronic inflammatory arthritis.

Apoptosis plays an essential role in the pathophysiologic processes of rheumatoid arthritis. A molecular probe that allows spatiotemporal observation of apoptosis in vitro, in vivo, and ex vivo concomitantly would be useful to monitoring or predicting pathophysiologic stages. In this study we investigated whether cyclic apoptosis-targeting peptide-1 (CApoPep-1) can be used as an apoptosis imaging probe in inflammatory arthritis. We tested the utility of CApoPep-1 for detecting apoptotic immune cells in vitro and ex vivo using flow cytometry and immunofluorescence. The feasibility of visualizing and quantifying apoptosis using this probe was evaluated in a murine collagen-induced arthritis (CIA) model, especially after treatment. CApoPep-1 peptide may successfully replace Annexin V for in vitro and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for ex vivo in the measurement of apoptotic cells, thus function as a sensitive probe enough to be used clinically. In vivo imaging in CIA mice revealed that CApoPep-1 had 42.9 times higher fluorescence intensity than Annexin V for apoptosis quantification. Furthermore, it may be used as an imaging probe for early detection of apoptotic response in situ after treatment. The CApoPep-1 signal was mostly co-localized with the TUNEL signal (69.6% of TUNEL+ cells) in defined cell populations in joint tissues of CIA mice. These results demonstrate that CApoPep-1 is sufficiently sensitive to be used as an apoptosis imaging probe for multipurpose applications which could detect the same target across in vitro, in vivo, to ex vivo in inflammatory arthritis.

A Pharmacological Strategy Using Stemofoline for more Efficacious Chemotherapeutic Treatments Against Human Multidrug Resistant Leukemic Cells

Our previous study reported that stemofoline (STF) exhibited a synergistic effect with chemotherapeutic drugs in human multidrug-resistant (MDR) leukemic cells (K526/Adr) by inhibiting the function of P-glycoprotein, which is a membrane transporter that is overexpressed in several types of MDR cancers. This study further investigated the effects of a combination treatment of STF and doxorubicin (DOX) in vitro and in vivo. The combination treatment of 50 mg/kg of STF strongly enhanced the anti-tumor activity of DOX in SCID-beige mice bearing K562/Adr xenografts without additional toxicity when compared to the single treatment groups. Additionally, an examination of the proliferation markers (Ki67) and the apoptotic marker (TUNEL) in tumor tissues in each group revealed that the combination therapy significantly reduced Ki67 positive cells and increased apoptotic cells. From the in vitro experiments we also found that this combination treatment dramatically induced G1 and G2M arrest in K562/Adr when compared to a single treatment of DOX. STF treatment alone did not show any cytotoxic effect to the cells. These results suggest that the accumulation of DOX enhanced by STF was sufficient to induce cell cycle arrest in K562/Adr. These findings support our previous in vitro data and indicate the possibility of developing STF as an adjuvant therapy in cancer treatments.

Protective effects of Kangxian ruangan capsule against nonalcoholic fatty liver disease fibrosis in rats induced by MCD diet.

Kangxian ruangan (KXRG) capsule is a classical formula containing various herbals that play a vital role of replenishing spleen and warming Yang. Traditional Chinese medicine believes that insufficiency of the spleen, damp-heat, and phlegm and stasis are the key factors to nonalcoholic fatty liver disease (NAFLD). The objective of this study was to investigate the effects of KXRG capsule on NAFLD fibrosis rats induced by MCD diet. The liver functions (ALT, AST and GGT) and levels of blood lipids (CHOL and TG) in each treatment group rats were significantly decreased, especially those in H-KXRG group. At the same time, the KXRG capsule alleviated the inflammatory response, histopathological changes and liver fibrosis of NAFLD fibrosis rats. In addition, the apoptosis of liver cells induced by diet was obvious via TUNEL staining. However, KXRG capsule reversed that negative change. Moreover, the levels of pro-apoptotic proteins (Caspase 3, 8, 9 and Bax) were reduced by exposure to KXRG capsule, except that the anti-apoptotic proteins (Bcl-2 and Bcl-XL) were elevated. In conclusion, KXRG relieved the progression of NAFLD fibrosis via maintaining the balance of TNF-α/IL-10 further relieving the inflammatory reaction, and regulating the balance of Bcl-2/Bax or Bcl-XL/Bax in a positive direction further activating damaged hepatocytes.

Protective effect of human umbilical cord mesenchymal stem cell exosomes on preserving the morphology and angiogenesis of placenta in rats with preeclampsia.

AIM: This study aims to investigate the effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Ex) on placental tissue and angiogenesis in rats with preeclampsia (PE). METHOD: The expression of MSC surface markers were identified by flow cytometry. Alizarin red staining and oil red O staining were used to examine osteogenic and adipogenic differentiation of hucMSCs. Western blotting was used to determine expressions of CD63 and CD81 in hucMSC-Ex. PE rat models were established using endothelial nitric oxide synthase, eNOS)NG-Nitro-l-arginine Methyl Ester, which were then treated with exosome (Exo) of low dosage (L-Exo), Exo of medium dosage (M-Exo) and Exo of high dosage (H-Exo). The blood pressure at the 15d, 17d and 19d of pregnancy and 24-h urinary protein were measured. TUNEL staining and immunohistochemistry were applied to detect the cell apoptosis and micro-vascular density (MVD) in placental tissues, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to examine serum levels of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase receptor-1 (sFlt1). RESULTS: In vitro cultured hucMSCs showed expression of MSC surface markers (CD29, CD90 and CD105), and no expression of CD34 and CD45. Besides, the isolated exosomes expressed the exosome markers (CD63 and CD81). In response to the treatment of L-Exo, M-Exo and H-Exo, the blood pressure of PE rat models on the 17 d and the 19 d as well as the 24-h urinary protein were substantially decreased. Moreover, at the 21 d, PE rat models treated with L-Exo, M-Exo and H-Exo exhibited an increase in the number and quality of fetuses, placenta quality, MVD and VEGF expression, but substantial decreased cell apoptosis and expression of sFlt1. The influence of Exos was exerted in a dosage dependent manner. CONCLUSION: hucMSC-Ex, in a dose-dependent manner, can improve the morphology of placental tissue in rats with PE, by inhibiting cell apoptosis and promoting angiogenesis in placental tissue.

Shengmai injection reduces apoptosis and enhances angiogenesis after myocardial ischaemia and reperfusion injury in rats.

OBJECTIVES: To investigate whether Shengmai injection (SMI) helps to improve cardiac function and enhances angiogenesis after myocardial ischaemia reperfusion injury (MIRI). A rat model of MIRI was created via occlusion of the left anterior descending coronary artery for 30 min, followed by 3 days or 7 days of reperfusion (n = 6 each group). BACKGROUNDS: SMI is widely used for the treatment of myocardial infarction. The mechanism underlying the effect on cardiac function is not known and whether SMI has any effects on angiogenesis during treatment of MIRI is not clear. RESULTS AND CONCLUSION: Echocardiography showed that SMI improved the left ventricular ejection fraction (LVEF) in the rat model of MIRI. TUNEL staining indicated that SMI decreased the myocardial apoptosis rate after MIRI. This result may be related to the increase of Bcl-2 expression in the SMI group and a reduction in Bax and caspase 3 expression, as determined by immunohistochemical staining. Small vessels (<60 µm in diameter) of the heart of rats in the group treated with SMI were denser (more numerous) than those in the heart of rats in the other groups. Real-time PCR indicated that the SMI-driven reduction in apoptosis was associated with a change in the ratio of Bcl-2 to Bax expression, and treatment-induced angiogenesis was associated with enhanced vascular endothelial growth factor A (VEGF) expression. We elucidated that SMI promotes angiogenesis, which is important for the development of cardiac remodelling after MIRI.

Qianlongtong Inhibits Proliferation and Induces Apoptosis of Hyperplastic Prostate Cells.

Qianlongtong is a compound made from traditional Chinese herbs and it has proven to be very effective to treat patients with benign prostate hypertrophy. However, its mechanism is still unknown. This study is designed to investigate the effect of Qianlongtong on proliferation and apoptosis of hyperplastic prostate cells. Flow cytometry (FCM) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to assess proliferation and apoptosis of hyperplastic prostate cells in the following groups: control group, tamoxifen group, and groups with low, moderate, and high dosage of Qianlongtong. Reverse transcription-polymerase chain reaction analysis was used to investigate the underlying mechanisms for increased apoptosis. Cells treated with Qianlongtong were mainly blocked in the G0/G1 phase. The apoptotic index of each group was significantly higher than that in the control group. The apoptotic index in the high- and moderate-dosage groups was similar to that in the tamoxifen group. The high- and moderate-dosage groups had lower Bcl-2 and higher Bax messenger RNA (mRNA) levels compared with the control group. Qianlongtong inhibits proliferation and promotes the apoptosis of hyperplastic prostate cells.

Valproic acid attenuates inflammation of optic nerve and apoptosis of retinal ganglion cells in a rat model of optic neuritis.

AIMS: Optic neuritis (ON) is an inflammatory disease of the optic nerve, which often occurs in patients with multiple sclerosis (MS) and leads to retinal ganglion cell (RGC) death and even severe visual loss. Valproic acid (VPA) is a short-chain branched fatty acid with anti-epileptic, neuro-protective and anti-inflammatory effects. Here, we examined the effects of VPA in experimental autoimmune encephalomyelitis (EAE) rats and explored the underlying mechanisms. MAIN METHODS: EAE was induced by subcutaneous injection with myelin basic protein, emulsified with complete Freund's adjuvant and Mycobacterium tuberculosis H37Ra into the Lewis rats. Subsequently, animals in the VPA groups were treated orally with VPA (250 or 500 mg/kg) once a day for 13 days. KEY FINDINGS: VPA treatment significantly attenuated inflammation and microgliosis in optic nerve in EAE-ON rats, as evidenced by the decrease in the mRNA levels of interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-17, and inducible nitric oxide synthase (iNOS), the suppression in nuclear factor (NF)-κB signal pathway as well as the down-regulation of CD11b expression in optic nerve. Additionally, the apoptotic RGCs were remarkably increased in the EAE retina, which was inhibited by VPA treatment. Consistent with the TUNEL staining, VPA administration also obviously suppressed the ratio of Bax: Bcl-2 and the expression of cleaved caspase-3 and PARP in optic nerve in EAE rats. SIGNIFICANCE: Our findings demonstrated that VPA treatment could prevent inflammation responses and RGC apoptosis in optic nerve in EAE-ON rats, suggesting that VPA may be available for optic nerve protection during ON.

TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats.

OBJECTIVE: to evaluate the influence of Tumor Necrosis Factor alpha (TNF-α) and apoptosis in rats with DM treated with chamomile extract or triamcinolone. MATERIAL AND METHODS: Wistar male rats (210.0±4.2 g) were divided into five groups: negative control group (NCG) without diabetes; positive control group (PCG) with DM (alloxan, 45 mg/kg); and groups treated with chamomile extract (normoglycemic= NCG group and diabetic= DCG group) and with triamcinolone (TG). Traumatic ulcers were performed on all animals that received topical triamcinolone, chamomile extract or saline 12/12 hours for ten days. RESULTS: On days five and ten the animals were euthanized and the ulcers were analyzed by light microscopy, TUNEL assay, and immunohistochemically (TNF-α). The NCG (p=0.0062), PCG (p=0.0285), NCG (p=0.0041), and DCG (p<0.0001) groups were completely healed on the 10th day, however, there was no healing on the TG (p=0.5127) group. The TNF-α expression showed a significant reduction from the 5th to the 10th day in NCG (p=0.0266) and DCG (p=0.0062). In connective tissue, the TUNEL assay showed a significant reduction in the number of positive cells in NCG (p=0.0273) and CNG (p=0.0469) and in the epithelium only in CDG (p=0.0320). CONCLUSIONS: Chamomile extract can optimize the healing of traumatic oral ulcers in diabetic rats through the reduction of apoptosis in the epithelium and TNF-α expression.

Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study.

Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis.

TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats

ABSTRACT Diabetes mellitus (DM) is a disease associated with delayed wound healing of oral ulcers by increased expression of proinflammatory cytokines and cellular apoptosis. Objective to evaluate the influence of Tumor Necrosis Factor alpha (TNF-α) and apoptosis in rats with DM treated with chamomile extract or triamcinolone. Material and Methods Wistar male rats (210.0±4.2 g) were divided into five groups: negative control group (NCG) without diabetes; positive control group (PCG) with DM (alloxan, 45 mg/kg); and groups treated with chamomile extract (normoglycemic= NCG group and diabetic= DCG group) and with triamcinolone (TG). Traumatic ulcers were performed on all animals that received topical triamcinolone, chamomile extract or saline 12/12 hours for ten days. Results On days five and ten the animals were euthanized and the ulcers were analyzed by light microscopy, TUNEL assay, and immunohistochemically (TNF-α). The NCG (p=0.0062), PCG (p=0.0285), NCG (p=0.0041), and DCG (p<0.0001) groups were completely healed on the 10th day, however, there was no healing on the TG (p=0.5127) group. The TNF-α expression showed a significant reduction from the 5th to the 10th day in NCG (p=0.0266) and DCG (p=0.0062). In connective tissue, the TUNEL assay showed a significant reduction in the number of positive cells in NCG (p=0.0273) and CNG (p=0.0469) and in the epithelium only in CDG (p=0.0320). Conclusions Chamomile extract can optimize the healing of traumatic oral ulcers in diabetic rats through the reduction of apoptosis in the epithelium and TNF-α expression.

Effects of Erlong Zuoci decoction on the age-related hearing loss in C57BL/6J mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Erlong Zuoci decoction (ELZCD), a typical traditional Chinese medicine (TCM) prescription, has long been clinically used in treatment of deafness and tinnitus with the syndrome of "kidney yin deficiency". However, there are few studies to investigate its pharmacological mechanisms. Until now, there is not report about its effects on the age-related hearing loss (ARHL). AIM OF STUDY: The present study was conducted to observe the effects of ELZCD on the ARHL in C57BL/6J mice and explore the mechanisms. MATERIALS AND METHODS: ELZCD was fed to C57BL/6J mice from 3 months to 6 months in ELZCD group as a dose of 6g/kg/d. And the same volume of saline was fed to mice in ARHL group. 3-months-old C57BL/6J mice were used as control group. High performance liquid chromatography (HPLC) was used for the quality control of ELZCD. Auditory brainstem response (ABR) was used to assess the hearing function of mice. The morphologic changes were observed by hematoxylin eosin (HE) staining. Apoptosis was tested by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. Mitochondrial damage was detected by transmission electron microscopy (TEM). Quantitative RT-PCR (qRT-PCR) was used to observe the mRNA expression of p53 and Bak. Fluorescence immunohistochemical technique was used to test the protein expression of p53 and Bak. RESULTS: The hearing threshold of ARHL group was higher than that of control group (P<0.001) and ELZCD decreased the rise of hearing threshold levels of ARHL mice (P<0.001), which suggested ELZCD inhibited the hearing loss of ARHL mice. HE staining showed that ELZCD decreased the spiral ganglion (SG) cell damage and loss in ARHL. TUNEL test showed that the apoptotic SG cells increased in ARHL group compared to control group and decreased in ELZCD group compared to ARHL group. TEM observation showed that mitochondrial damage was obvious in SG cells of ARHL group and ELZCD inhibited the mitochondrial damage. The qRT-PCR results showed that the mRNA expression of p53 and Bak in ARHL group increased compared to that of control group (P<0.05), and ELZCD reduced the elevated mRNA expression levels of p53 and Bak (P<0.01, P<0.05). In addition, ELZCD inhibited the increased proteins expression (green fluorescence) of p53 and Bak. CONCLUSION: The results demonstrated that ELZCD prevented ARHL in C57BL/6J mice and p53/Bak-mediated mitochondrial apoptosis of SG cells might be involved in the mechanisms.

Amelioration of acute myocardial infarction by saponins from flower buds of Panax notoginseng via pro-angiogenesis and anti-apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax notoginseng is traditionally used as an anti-hemorrhagic agent to promote blood circulation without causing "congealed" blood. Furthermore, the flower of P. notoginseng is a popular, traditional medicine taken daily for the preventing of hypertension and for reducing blood cholesterol profiles. Besides, the flower of P. notoginseng contains a higher level of saponins, particularly protopanaxadiol-type ginsenosides, as compared to the root. However, detailed pharmacological studies on this flower have rarely been conducted. MATERIAL AND METHODS: In this study, the saponins extracted from the flower of P. notoginseng (FS) were examined on the endothelial cell migration assay, chemically induced vascular insufficiency model in zebrafish larvae and myocardial infraction (MI) model in rats, for determination of their pro-angiogenic and therapeutic effects on MI treatment. RESULTS: Our results demonstrate that FS significantly promoted VEGF-induced migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels (ISV) in a chemically induced vascular insufficiency model of zebrafish larvae. When compared to MI group, two weeks post-treatment of FS (25-50mg/kg/day) induced approximately 3-fold upregulation of VEGF mRNA expression and a concomitant increase in blood vessel density in the peri-infarct area of the heart. Moreover, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. CONCLUSIONS: The pro-angiogenic effects of FS demonstrated in in vitro and in vivo experimental models suggest that the purified saponin preparation from flowers of P. notoginseng may potentially provide preventive and therapeutic agent for cardiovascular diseases.

Vinyl chloride monomer (VCM) induces high occurrence of neural tube defects in embryonic mouse brain during neurulation.

The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.

Microscopic analysis of chromatin localization and dynamics in C. elegans.

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.

Antenatal supplementation of taurine for protection of fetal rat brain with intrauterine growth restriction from injury by reducing neuronal apoptosis.

This study aimed to investigate whether antenatal taurine can reduce neuronal apoptosis in fetal rat brains with intrauterine growth restriction (IUGR) and its possible mechanisms. A total of 15 pregnant rats were randomly divided into the following three groups: control, IUGR, and IUGR+ antenatal taurine supplements. Neuronal apoptosis was detected using transferase-mediated dUTP biotin nick end-labeling (TUNEL); the expression of Bcl-2, Bax, and caspase-3 mRNA and proteins was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. In IUGR groups, the results were as follows: (1) the expression of Bcl-2 decreased whereas the expression of Bax increased, accordingly, the ratio of Bcl-2/Bax decreased, (2) the expression of caspase-3 increased significantly, and (3) apoptotic neuron counts in IUGR groups was significantly increased compared with controls. In taurine supplement groups, the results were as follows: (1) the expression of Bcl-2 increased whereas the expression of Bax decreased, accordingly, the ratio of Bcl-2/Bax increased, (2) the expression of caspase-3 in fetal rat cerebral cortex tissues decreased significantly, and (3) the number of apoptotic neurons was significantly decreased compared with IUGR groups. In addition, the changes in the expression of Bcl-2, Bax, and caspase-3 mRNA and protein were correlated. So we concluded that antenatal supplementation of taurine may reduce neuronal apoptosis in IUGR fetal rats via up-regulating the ratio of Bcl-2/Bax and down-regulating the expression of caspase-3.

Magnetic stent hyperthermia for esophageal cancer: an in vitro investigation in the ECA-109 cell line.

Magnetic stent hyperthermia (MSH) is a novel approach for targeted thermotherapy for esophageal cancer, which is based on the mechanism that inductive heat can be generated by the esophageal stent upon exposure under an alternative magnetic field (AMF). A positive effect of MSH on esophageal cancer has been demonstrated, however, there is no study on the in vitro effects of heating treatment or of the effects of AMF exposure on human esophageal cancer cells. This study aimed to investigate the effect of MSH and of AMF exposure in esophageal cancer cells. Inductive heating characteristics of esophageal stents were assessed by exposing the stents under AMF. A rather rapid temperature rise of the Ni-Ti stent when subjected to AMF exposure was observed and the desired hyperthermic temperature could be controlled by adjusting the field parameter of the AMF. Human esophageal squamous carcinoma (ESCC) ECA-109 cells were divided into four groups: the control group, the water-bath heating group, the MSH group and the AMF exposure group. Hyperthermic temperatures were 43, 48 and 53˚C and the treatment time was in the range of 5-30 min. The MTT assay, apoptotic analysis and TUNEL staining were applied in the current investigation. Exposure of ECA-109 cells under AMF with a field intensity of 50 to 110 kA/m had negligible effect on cell viability, cell necrosis and apoptosis. Hyperthermia had a remarkable inhibitory effect on the cell viability and the effect was dependent on the thermal dose (temperature and time). The optimal thermal dose of MSH for ECA-109 cells was 48˚C for 20-30 min. The study also elucidated that there was a difference in the effects on cell necrosis and apoptosis between the heating mode of water bath and MSH. The data suggest that MSH may have clinical significance for esophageal cancer treatment.

Inhibition of inflammation and oxidative stress by Angelica dahuricae radix extract decreases apoptotic cell death and improves functional recovery after spinal cord injury.

Inflammation and oxidative stress play major roles in the pathogenesis after spinal cord injury (SCI). Here, we examined the neuroprotective effects of Angelica dahuricae radix (ADR) extract after SCI. ADR extract significantly decreased the levels of proinflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in a lipopolysaccharide (LPS)-activated microglial cell line, BV2 cells. ADR extract also significantly alleviated the level of reactive oxygen species in LPS-activated BV2 cells. To examine the neuroprotective effect of ADR extract after SCI, spinally injured rats were administered ADR extract orally at a dose of 100 mg/kg for 14 days. ADR extract treatment significantly reduced the levels of TNF-α, IL-1ß, IL-6, iNOS, and COX-2. The levels of superoxide anion (O(2·)(-)) and protein nitration were also significantly decreased by ADR extract. In addition, ADR extract inhibited p38 mitogen-activated protein kinase activation and pronerve growth factor expression in microglia after SCI. Furthermore, ADR extract significantly inhibited caspase-3 activation following apoptotic cell death of neurons and oligodendrocytes, thereby improving functional recovery after injury. Thus, our data suggest that ADR extract provides neuroprotection by alleviating inflammation and oxidative stress and can be used as an orally administered therapeutic agent for acute SCI.

Catechin hydrate inhibits proliferation and mediates apoptosis of SiHa human cervical cancer cells.

Catechin hydrate (CH), one of the chemical compounds in green tea, has been shown to inhibit tumor growth. Green tea possesses anticancer potential and is one of the most commonly used herbal medicines worldwide. In this study, we sought to characterize the DNA damage and downstream genes targeted by CH extracts using SiHa human cervical cancer cells. The efficacy of CH in killing cervical cancer cells in vitro was investigated in this study to determine whether CH possesses anticancer potential and could be developed as a therapeutic agent for cervical cancer upon further investigation. To scientifically validate the anticancer activities of CH on cervical cancer, CH was tested for its cytotoxic and growth-inhibition properties, specifically the induction of apoptosis in SiHa cervical cancer cells. CH showed a 50% inhibition of SiHa human cervical cancer cells at a concentration of 196.07 µg/mL at 24h. CH induced the several folds increase of caspase-3, -8, and -9 after 24h and 48 h; the increase of these genes may be involved in the induction of apoptosis. The analysis of apoptosis by DeadEnd terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL) assay was used to further confirm that CH induced apoptosis. The results suggested that CH has the potential to benefit cervical cancer prevention. This is the first report that shows the possible mechanism of the anti-proliferative effects of CH in the prevention of cervical cancer in cell culture models. CH, either in its original form or in combination with other anticancer drugs, could potentially be an alternative medicine for cervical cancer. Further study may increase our understanding of the mechanism by which CH has an effect on cervical cancer therapy.

Protective effects of diosmetin extracted from Galium verum L. on the thymus of U14-bearing mice.

Diosmetin (DGVL) extracted from the traditional Chinese herb Galium verum L. has been found to have anticancer activity. In this study, the effects of DGVL on the thymus of U14-bearing mice were investigated. Using flow cytometry, peripheral blood lymphocytes were characterized based on the expression of surface markers for T helper cells (CD4(+)) and T suppressor cells (CD8(+)). Serum levels of tumor necrosis factor α (TNF-α), interleukin-2 (IL-2), IL-10, and transforming growth factor ß1 (TGF-ß1) and a cell proliferation assay were determined with an enzyme-linked immunosorbent assay. The expression of Fas and Fas ligand (FasL) on the thymus was determined by Western blotting. Our results showed that DGVL inhibited tumor growth and significantly increased the thymus weight compared with the control. Also, DGVL elevated serum levels of IL-2 and significantly reduced levels of TNF-α, TGF-ß1, and IL-10 in a dose-dependent manner. Histological study and terminal dUTP nick end labeling staining results showed that DGVL protected thymus tissue against the onslaught of tumor growth by inhibiting thymus lymphocyte apoptosis. The cell proliferation assay revealed that DGVL might promote more thymus lymphocytes towards proliferation. Furthermore, the ratio of CD4(+)/CD8(+) T lymphocytes was significantly increased from 0.69 to 2.29 by treatment with DGVL. Immunoblotting analyses revealed that the expression of Fas and FasL on the thymus was lower in mice in the DGVL treatment group than in the control mice. In conclusion, DGVL can inhibit tumor growth and protect tumor-induced apoptosis of the thymus, and the mechanism is closely associated with reduced cell death in the thymus and a Fas-FasL-dependent pathway.

Iron accumulation and DNA damage in a pig model of intracerebral hemorrhage.

Cerebral iron overload causes brain injury after intracerebral hemorrhage (ICH) in rats and pigs. The current study examined whether an iron chelator, deferoxamine, can reduce ICH-induced DNA damage in pigs. Pigs received an injection of autologous blood into the right frontal lobe. Deferoxamine (50 mg/kg, i.m.) or vehicle was given 2 h after ICH and then every 12 h up to 7 days. Animals were killed at day 3 or day 7 after ICH to examine iron accumulation and DNA damage. We found that ICH resulted in the development of a reddish perihematomal zone, with iron accumulation and DNA damage within that zone. Deferoxamine treatment reduced the perihematomal reddish zone, and the number of Perls' (p<0.01) and TUNEL (p<0.01) positive cells. In conclusion, iron accumulates in the perihematomal zone and causes DNA damage. Systemic deferoxamine treatment reduces ICH-induced iron overload and DNA damage in pigs.