The potential role of hydrogen sulfide in cancer cell apoptosis.

For a long time, hydrogen sulfide (H2S) has been considered a toxic compound, but recent studies have found that H2S is the third gaseous signaling molecule which plays a vital role in physiological and pathological conditions. Currently, a large number of studies have shown that H2S mediates apoptosis through multiple signaling pathways to participate in cancer occurrence and development, for example, PI3K/Akt/mTOR and MAPK signaling pathways. Therefore, the regulation of the production and metabolism of H2S to mediate the apoptotic process of cancer cells may improve the effectiveness of cancer treatment. In this review, the role and mechanism of H2S in cancer cell apoptosis in mammals are summarized.

IL-33/ST2L signaling alleviates diabetic nephropathy by regulating endoplasmic reticulum stress and apoptosis.

OBJECTIVE: Diabetic nephropathy (DN) is a serious chronic complication of diabetes mellitus (DM). Endoplasmic reticulum (ER) stress is an important factor in the regulation of pathological processes in DN, and excessive ER stress can lead to apoptosis. Although the IL-33/ST2 axis is known to be involved in diabetic kidney disease or related nephropathy, its role and molecular mechanisms remain poorly understood in terms of DN. The purpose of this study was to investigate the effects of IL-33/ST2 signaling on DN and to characterize the roles that ER stress and apoptosis play in DN. METHODS: To investigate this study, mice were randomly assigned into DN (induced by 0.1% STZ) and Control groups. Biochemical indices (FBG, BUN, UPR, UCE) were measured in serum and urine samples to reflect blood glucose and kidney damage. Quantitative real-time PCR, western blot, and immunofluorescence were used to assess gene and protein expression of the IL-33/ST2 axis and ER stress relative signaling molecule. Apoptosis was analyzed by flow cytometry. RESULTS: IL-33 levels are significantly increased in the kidneys of patients and mice with DN. Double immunofluorescence staining showed that IL-33 colocalized with CD31-positive endothelial cells. Treatment with IL-33 attenuated kidney injury in Streptozotocin (STZ)-treated mice. In vitro, we showed that IL-33 attenuated ER stress and apoptosis in glomerular endothelial cells. However, sST2 treatment significantly reversed these effects of IL-33. CONCLUSION: Together, these data suggest that IL-33/ST2 signaling mitigates STZ-induced renal damage, partly at least, by suppressing ER stress and apoptosis. Therefore, IL-33 may be an effective therapeutic target in DN.

In-depth transcriptome characterization uncovers distinct gene family expansions for Cupressus gigantea important to this long-lived species' adaptability to environmental cues.

BACKGROUND: Cupressus gigantea, a rare and endangered tree species with remarkable medicinal value, is endemic to the Tibetan Plateau. Yet, little is known about the underlying genetics of the unique ecological adaptability of this extremely long-lived conifer with a large genome size. Here, we present its first de novo and multi-tissue transcriptome in-depth characterization. RESULTS: We performed Illumina paired-end sequencing and RNA libraries assembly derived from terminal buds, male and female strobili, biennial leaves, and cambium tissues taken from adult C. gigantea. In total, large-scale high-quality reads were assembled into 101,092 unigenes, with an average sequence length of 1029 bp, and 6848 unigenes (6.77%) were mapped against the KEGG databases to identify 292 pathways. A core set of 41,373 genes belonging to 2412 orthologous gene families shared between C. gigantea and nine other plants was revealed. In addition, we identified 2515 small to larger-size gene families containing in total 9223 genes specific to C. gigantea, and enriched for gene ontologies relating to biotic interactions. We identified an important terpene synthases gene family expansion with its 121 putative members. CONCLUSIONS: This study presents the first comprehensive transcriptome characterization of C. gigantea. Our results will facilitate functional genomic studies to support genetic improvement and conservation programs for this endangered conifer.

Directly reprogramming fibroblasts into adipogenic, neurogenic and hepatogenic differentiation lineages by defined factors.

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types represents a great potential technology for regenerative medicine. In the present study, the potential of key developmental adipogenic, neurogenic and hepatogenic regulators to reprogram human fibroblasts into adipocytes, neurocytes and hepatocytes was investigated. The results demonstrated that direct reprogramming of octamer-binding transcription factor 4 (Oct4) and CCAAT-enhancer-binding protein (C/EBP)ß activated C/EBPα and peroxisome proliferator-activated receptor-γ expression, inducing the conversion of fibroblasts into adipocytes. Similarly, direct reprogramming of the transcription factors sex determining region-box 2, trans-acting T-cell specific transcription factor (GATA-3) and neurogenic differentiation 1 in fibroblasts may induce neurogenic differentiation through hemagglutinating virus of Japan envelope (HVJ-E) transfection. Moreover, hepatogenic differentiation was induced by combining the direct reprogramming of Oct4, GATA-3, hepatocyte nuclear factor 1 homeobox α and forkhead box protein A2 in fibroblasts. These results demonstrate that specific transcription factors and reprogramming factors are able to directly reprogram fibroblasts into adipogenic, neurogenic and hepatogenic differentiation lineages by HVJ-E transfection.

Bone marrow-derived cells response in proximal regions of nerves after peripheral nerve injury.

In recent years, bone marrow-derived cells have been found to be crucial for peripheral nerve regeneration. After traumatic peripheral nerve injury, bone marrow-derived macrophages quickly infiltrate into the distal regions of nerves. To explore the changes caused by bone marrow-derived cells within the proximal regions of the nerves, sciatic nerves of chimeric mice carrying bone marrow cells expressing green fluorescent protein were crushed to observe the infiltration of invading bone marrow-derived cells. Seven days after surgery, abundant bone marrow-derived cells had infiltrated into the damaged proximal nerve segments. The numbers of these cells increased to a peak at 2 weeks and then gradually returned to normal levels within 30 weeks. Through immunofluorescence staining, many of these cells were identified as macrophages, and they showed a similar infiltration tendency toward distal nerve segments. However, fewer cells infiltrated proximal segments than distal nerve segments. In conclusion, these findings suggest that bone marrow-derived cells response not only occurs within the distal nerve segments but may also take place within the proximal segments of nerve tissues after nerve injury.

SOCS3 treatment prevents the development of alopecia areata by inhibiting CD8+ T cell-mediated autoimmune destruction.

Alopecia areata is one of the most common autoimmune diseases resulting from T cell-mediated damage of hair follicles. CD8+ T cells infiltrate hair follicles and are responsible for destruction of hair follicles. However the underlying mechanisms for hair loss remain still obscure. In the present study, we identified that suppressor of cytokine signaling-3 (SOCS3), a classical inhibitor of cytokine signaling, significantly inhibits CD8+T cell maturation, interferon-γ (IFN-γ) production and alopecia areata. SOCS3 is downregulated in the skin of alopecia areata patients and murine autoimmune alopecia model. Furthermore, SOCS3 treatment prevents the development of alopecia areata in the graft model. SOCS3 decreases the CD44high CD62Llow effector memory CD8+ T cells, resulting in the decrease of IFN-γ production. The expression of Fas and major histocompatibility complex-1 (MHC I) is upregulated in skin from C3H/HeJ alopecia areata mice, and this increase is suppressed by SOCS3. The SOCS3 level is negative correlation with the Fas and MHC I level in patients with alopecia areata. These results suggest that SOCS3 treatment may be an effective strategy to treat autoimmune alopecia as well as to more generally prevent cytokine-dependent tissue destruction in inflammatory diseases.

[Assay of Tetramethylpyrazine in Szechwan Lovage Rhizome and Cnidium Rhizome by HPLC-DAD-MS].

OBJECTIVE: To quantity the amount of tetramethylpyrazine in Szechwan Lovage Rhizome (Chuanxiong, the rhizome of Ligusticum chuanxiong Hort., CX) and Cnidium Rhizome(Japanese Chuanxiong, the rhizome of Cnidium officinale Makino, JCX) for quality assessment. METHODS: An HPLC-DAD-MS technique was employed to detect tetramethylpyrazine in 27 CX and 10 JCX samples. Tetramethylpyrazine was separated on a Waters Symmetry C,, column (250 mm x 4. 6 mm, 5 µm). The mobile phase was methanol-acetonitrile-water(27: 1: 72) at a flow rate of 1. 0 mL/min. The column temperature was 35 °C. DAD detection wavelength was 280 nm, while electrospray ionization detector was set at positive mode to collect MS spectrum. RESULTS: In the total of 37 herb samples, 11 samples were found to contain tetramethylpyrazine with the mean amount of 2. 19 µg/g(n = 11). 6 of 27 CX samples and 5 of 10 JCX sample were found the existence of tetramethylpyrazine with the amount of 0. 60 - 11. 75 µg and 0. 61 - 3. 05 µg/g,respectively. The correlation was not found between tetramethylpyrazine and the cultivation area, morphological character, processing or storage method for CX and JCX samples. It was possible that tetramethylpyrazine resulted from the microbes in soil. CONCLUSION: The developed method is accurate to quantify tetramethylpyrazine in CX and JCX herbs. Both the two herbs indeed contain tetramethylpyrazine, but it is not suitable to be a chemical marker to assess the quality of CX and JCX owing to low content.

Specific marker expression and cell state of Schwann cells during culture in vitro.

Schwann cells (SCs) in animals exist in different developmental stages or wound repair phases, distinguished mainly by the expression of SC-specific markers. No study has yet determined SC state under in vitro culture conditions, and the specific markers expressed in SC are obscure as well. In this study, we harvested sciatic nerves from newborn mice and isolated SCs by an enzyme-digestion method, then we examined the expression profiles of ten markers (S100, p75NTR, Sox10, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ) at both the RNA and protein levels in in vitro mouse SCs and speculated their relation with in vivo SC stages. We assayed RNA and protein levels of SC specific markers by immunofluorescence, Western Blot, and real-time quantitative RT-PCR. The results show that the expression of most markers (S100, p75NTR, GAP43, NCAM, Krox20, Oct6, MBP and MPZ) was not detectable in all of early stage cultured SCs. The expression of transcription factors Sox10 and Sox2 was, however, detectable in all SCs. After 8 days, the positive expression rate of all markers except GAP43 and Oct6 was almost 100%.These results indicates Sox10 is a necessary marker for SC identification, while S100 is not reliable. SCs cultured in vitro express Sox2, P75NTR, NCAM, GAP43, Oct6, and MPZ, suggesting that they are similar to in vivo undifferentiated iSCs or dedifferentiated iSCs after nerve injury.

[Chemical change of chuanxiong raw materials during storage].

OBJECTIVE: To evaluate the chemical changes in Chuanxiong raw material (CX), the rhizome of Ligusticum chuanxiong Hort., during CX storage and further assess its quality variety. METHODS: Four CX samples were sealed and stored at ambient temperature in room for two years. These samples were quantified on the amounts of characteristic chemical compounds by HPLC-DAD-(APCI) MS techniques. RESULTS: Eight characteristic peaks in HPLC fingerprint were found to be good separation and assigned as vanillin (1), ferulic acid (2), senkyunolide I (3), senkyunolide H (4), coniferyl ferulate (5), senkyunolide A (6), Z-ligustilide (7) and levistolide A(8), respectively based on their on-line APCI-MS data and UV spectra. After CX being stored, compounds 1 - 4, and 8 were decreased by 44.4%, 52.1%, 37.6%, 52.8% and 47.5% (n = 4), respectively, whilst compounds 5 - 7 were increased by 59.1%, 40.1% and 47.5% (n = 4), respectively. CONCLUSION: Multiple chemical compounds are found to be changed during CX storage, which results in the variety of quality and therapeutic effect because most of the tested compounds have been demonstrated to be bioactive by pharmacological study and clinical trials. It is suggested that CX should be stored under dark, cool and dry condition.

Peripheral nerve repair with epimysium conduit.

Autologous tissues such as skeletal muscle have high biocampatibility and can effectively promote nerve regeneration compared to other biological and artificial materials; however, the reasonable and effective application of skeletal muscle requires further study. The purpose of this investigation was to assess the possibility of preparing a hollow nerve conduit, termed the epimysium conduit (EMC), using thin crimps of epimysium with skeletal muscle fibers and evaluate its effectiveness in repairing peripheral nerve defects. We prepared nerve conduits containing lumen with the external oblique muscle of the CAG-EFGP transgenic mice using microsurgical techniques for bridge repair of a 5-mm long sciatic nerve defect in wild-type mice. Systematic histological and functional assessments of the regenerated nerves were performed 8 and 12 weeks after surgery. EMC was found to effectively repair the sciatic nerve defect with significantly greater effectiveness than artificial conduits; however, the repair effect of EMC was lower than that of autologous nerve grafting for some parameters. In addition, our findings showed that some EMC-derived cell components migrated into the region of the regenerated nerves and contributed to reconstruction. Based on these findings, we conclude that a hollow conduit prepared with epimysium and a few skeletal muscle fibers is ideal for repairing peripheral nerve defects, and the cell components in the grafts contribute to nerve regeneration and structural remodeling, which provides an alternative option for the emergency primary repair of peripheral nerve defects in clinical practice.

[Comparison on content of ligustilides in different danggui samples].

Bioactivity of Danggui is linked to the content of ligustilide, but the relationship between ligustilide with herb shape, cultivating areas and plant species is still unknown. The relationship was investigated by quantifying on the amounts of Z-ligustilide and E-ligustilide by HPLC-DAD-MS method, and then comparing the content of ligustilides (the sum of Z-ligustilide and E-ligustilide) among forty-four various "Danggui" samples containing thirty Chinese Danggui (CDG), six Japanese Danggui (JDG), four Korea Danggui (KDG) and four European Danggui (EDG). Results showed that the content of ligustilides in CDG samples (Angelica sinensis) was in the range of 5.63-24.53 mg x g(-1) with the mean of 11.02 mg x g(-1) (n = 30). Ligustilides amounts were varied among samples cultivated in different areas in China, i. e. 13.90 mg x g(-1) (n = 6) in Yannan, 12.51 mg x g(-1) (n = 6) in Sichuan and 10.04 mg x g(-1) (n = 13) in Gansu. It was also found that ligustilides content was related to the shape, color and fragrance of herb, e. g. the relative larger amount of ligustilides was in the small main root, long rootlet and perfumed sample. Further, ligustilides contents were estimated to be 1.00 mg x g(-1) (n = 6) in JDG samples (A. acutiloba and A. acutiloba var. sugiyamae) and 2.78 mg x g(-1) (n = 2) in EDG samples (lovage root, Levisticum officinale). However, ligustilides could not be detected in the four KDG samples (A. gigas) and two EDG samples (angelica root, A. archangelica). It has been concluded that ligustilide is significant variant among plant species, which may result in the variety of bioactivity and therapeutic effect.

Ciliary neurotrophic factor-coated polylactic-polyglycolic acid chitosan nerve conduit promotes peripheral nerve regeneration in canine tibial nerve defect repair.

A variety of nerve conduits incorporated with chemical and biological factors have been developed to further stimulate nerve regeneration. Although most of the nerve guides in studies are basically limited to bridge a short gap of nerve defect in rat models, it is vital to evaluate effects of conduits on nerve regeneration over distance greater than 20 mm, or more clinically relevant nerve gap lengths in higher mammals. In this study, a poly(lactide-co-glycolide) (PLGA) nerve conduit, treated with pulsed plasma and coated with ciliary neurotrophic factor (CNTF) as well as chitosan, was used to repair 25-mm-long canine tibial nerve defects in eighteen cross-bred dogs. The canines were randomly divided into three groups (n = 6), a 25-mm segment of the tibial nerve was removed and replaced by a PLGA/chitosan-CNTF nerve conduit, PLGA/chitosan conduit and autologous nerve grafts were performed as the control. The results were evaluated by general observation, electromyogram testing, S-100 histological immunostaining, and image analysis at 3 months after operation. The histological results demonstrated that the PLGA/chitosan-CNTF conduits and PLGA/chitosan conduits were capable of leading the damaged axons through the lesioned area. Through the comparison of the three groups, the results in PLGA/chitosan-CNTF conduits group were better than that of PLGA/chitosan conduits group, while they were similar to autologous nerve grafts group. Therefore, CNTF-coated PLGA/chitosan nerve conduits could be an alternative artificial nerve conduit for nerve regeneration.

Purification of Schwann cells from adult rats by differential detachment.

Differential detachment by collagenase treatment is a new and efficient method for Schwann cell (SC) purification. As its effect on adult animals remains unclear, we have investigated the possibility of SC purification from adult rats. To avoid any systematic bias, Schwann cell purity before and after purification were compared by morphology, immunostaining of P75(NTR) and S100 and flow cytometric analysis. The final SC purities reached 99% as confirmed by three independent analyses SC purity and the cell yields were above 10(6) cells after two rounds of purification. The method of differential detachment is also suitable for SC purification in adult rats and could be useful for research and clinical applications.

Adenoviral transduction of hTGF-beta1 enhances the chondrogenesis of bone marrow derived stromal cells.

TGF-beta1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-beta1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-beta1 expressed and secreted more hTGF-beta1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-beta1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 +/- 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-beta1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-beta1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.

Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) are a widely researched adult stem cell population capable of differentiation into various lineages. Because many promising applications of tissue engineering require cell expansion following harvest and involve the treatment of diseases and conditions found in an aging population, the effect of donor age and ex vivo handling must be understood in order to develop clinical techniques and therapeutics based on these cells. Furthermore, there currently exists little understanding as to how these two factors may be influenced by one another. RESULTS: Differences in the adipogenic, chondrogenic, and osteogenic differentiation capacity of murine MSCs harvested from donor animals of different age and number of passages of these cells were observed. Cells from younger donors adhered to tissue culture polystyrene better and proliferated in greater number than those from older animals. Chondrogenic and osteogenic potential decreased with age for each group, and adipogenic differentiation decreased only in cells from the oldest donors. Significant decreases in differentiation potentials due to passage were observed as well for osteogenesis of BMSCs from the youngest donors and chondrogenesis of the cells from the oldest donors. CONCLUSION: Both increasing age and the number of passages have lineage dependent effects on BMSC differentiation potential. Furthermore, there is an obvious interplay between donor age and cell passage that in the future must be accounted for when developing cell-based therapies for clinical use.

Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment.

Schwann cell purification is usually difficult due to the contamination of fibroblasts, which often become a predominant cell type in Schwann cell culture in vitro. We have developed a novel and efficient method to enrich Schwann cells by differential detachment of two types of cells. In the culture, cells were treated with a multiplex collagenase and the Schwann cells were found to detach faster than fibroblasts and thus Schwann cells could be easily isolated. Within 5 days, Schwann cell purity could reach above 99%, which was confirmed by immunostaining characterization and flow cytometric analysis. In addition, this efficient method can reach a high cell yield after two rounds of differential detachment procedures and does not require antimitotic treatment or special equipment as often needed by other reported methods.