Syringic acid promotes cartilage extracellular matrix generation and attenuates osteoarthritic cartilage degradation by activating TGF-ß/Smad and inhibiting NF-κB signaling pathway.

Osteoarthritis (OA) is a common chronic degenerative disease which is characterized by the disruption of articular cartilage. Syringic acid (SA) is a phenolic compound with anti-inflammatory, antioxidant, and other effects including promoting osteogenesis. However, the effect of SA on OA has not yet been reported. Therefore, the purpose of our study was to investigate the effect and mechanism of SA on OA in a mouse model of medial meniscal destabilization. The expressions of genes were evaluated by qPCR or western blot or immunofluorescence. RNA-seq analysis was performed to examine gene transcription alterations in chondrocytes treated with SA. The effect of SA on OA was evaluated using destabilization of the medial meniscus model of mice. We found that SA had no obvious toxic effect on chondrocytes, while promoting the expressions of chondrogenesis-related marker genes. The results of RNA-seq analysis showed that extracellular matrix-receptor interaction and transforming growth factor-ß (TGF-ß) signaling pathways were enriched among the up-regulated genes by SA. Mechanistically, we demonstrated that SA transcriptionally activated Smad3. In addition, we found that SA inhibited the overproduction of lipopolysaccharide-induced inflammation-related cytokines including tumor necrosis factor-α and interleukin-1ß, as well as matrix metalloproteinase 3 and matrix metalloproteinase 13. The cell apoptosis and nuclear factor-kappa B (NF-κB) signaling were also inhibited by SA treatment. Most importantly, SA attenuated cartilage degradation in a mouse OA model. Taken together, our study demonstrated that SA could alleviate cartilage degradation in OA by activating the TGF-ß/Smad and inhibiting NF-κB signaling pathway.

The neuroprotective effects of Liuwei Dihuang medicine in the APP/PS1 mouse model are dependent on the PI3K/Akt signaling pathway.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that progressively impairs cognitive function and memory. The occurrence and development of Alzheimer's disease involves many processes. In response to the complex pathogenesis of AD, the Traditional Chinese medicine formula Liuwei Dihuang Pill (LWD) has been shown to improve the cognitive function of AD animal models. However, the active ingredients and mechanism of action of LWD have not been fully elucidated. In this study, network pharmacological analysis predicted 40 candidate compounds in LWD, acting on 227 potential targets, of which 185 were associated with AD. Through network pharmacological analysis, the mechanism of action of LWD therapy AD is related to the inhibition of inflammatory response, regulation of neuronal state, and autophagy. In this experiment, LWD was detected in the APP/PS1 transgenic mouse model. The objective was to observe the effects of LWD on hippocampal learning and memory ability, Aß clearance, autophagy and inflammatory response in APP/PS1 mice. The results showed that LWD improved long-term memory and working memory in APP/PS1 mice compared with the WT group. At the same time, LWD can increase the expression of hippocampal autophagy biomarkers, reduce the precipitation of Aß, and the activation of microglia and astrocytes. Its mechanism may be related to the regulation of the PI3K/Akt signaling pathway. Thus, we demonstrate for the first time that LWD has a neuroprotective effect on APP/PS1 mice and provide theoretical foundation for the development of a new clinical treatment for AD.

Identification of Interleukin-1-Beta Inhibitors in Gouty Arthritis Using an Integrated Approach Based on Network Pharmacology, Molecular Docking, and Cell Experiments.

Background: This study aimed to investigate the molecular mechanism of Tongfengding capsule (TFDC) in treating immune-inflammatory diseases of gouty arthritis (GA) and interleukin-1-beta (IL-1ß) inhibitors by using network pharmacology, molecular docking, and cell experiments. Methods: In this study, the compounds of TFDC and the potential inflammatory targets of GA were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Online Mendelian Inheritance in Man (OMIM), and GeneCards databases. The TFDC-GA-potential targets interaction network was accomplished by the STRING database. The TFDC-active compound-potential target-GA network was constructed using Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to further explore the GA mechanism and therapeutic effects of TFDC. Quantitative real-time PCR (qPCR) was used to verify whether the TFDC inhibited IL-1ß in GA. Molecular docking technology was used to analyze the optimal effective compounds from the TFDC for docking with IL-1ß. Result: 133 active compounds and 242 targets were screened from the TFDC, and 25 of the targets intersected with GA inflammatory targets, which were considered as potential therapeutic targets. Network pharmacological analysis showed that the TFDC active compounds such as quercetin, stigmasterol, betavulgarin, rutaecarpine, naringenin, dihydrochelerythrine, and dihydrosanguinarine had better correlation with GA inflammatory targets such as PTGS2, PTGS1, NOS2, SLC6A3, HTR3A, PPARG, MAPK14, RELA, MMP9, and MMP2. The immune-inflammatory signaling pathways of the active compounds for treating GA are IL-17 signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, HIF-1 signaling pathway, etc. The TFDC reduced IL-1ß mRNA expression in GA by qPCR. Molecular docking results suggested that rutaecarpine was the most appropriate natural IL-1ß inhibitor. Conclusion: Our findings provide an essential role and bases for further immune-inflammatory studies on the molecular mechanisms of TFDC and IL-1ß inhibitors development in GA.

Amino-Acid-Encoded Supramolecular Photothermal Nanomedicine for Enhanced Cancer Therapy.

Photothermal nanomedicine based on self-assembly of biological components, with excellent biosafety and customized performance, is vital significance for precision cancer therapy. However, the programmable design of photothermal nanomedicine remains extremely challenging due to the vulnerability and variability of noncovalent interactions governing supramolecular self-assembly. Herein, it is reported that amino acid encoding is a facile and potent means to design and construct supramolecular photothermal nanodrugs with controlled therapeutic activities. It is found that the amount and type of amino acid dominates the assembled nanostructures, structural stability, energy-conversion pathway, and therapeutic mechanism of the resulting nanodrugs. Two optimized nanodrugs are endowed with robust structural integrity against disassembly along with high photothermal conversion efficiency, efficient cellular internalization, and enhanced tumor accumulation, which result in more efficient tumor ablation. This work demonstrates that design based on amino acid encoding offers an unprecedented opportunity for the construction of remarkable photoactive nanomedicines toward cancer diagnostics and therapeutics.

Supramolecular cancer photoimmunotherapy based on precise peptide self-assembly design.

Combinational photoimmunotherapy (PIT) is considered to be an ideal strategy for the treatment of highly recurrent and metastatic cancer, because it can ablate the primary tumor and provide in situ an autologous tumor vaccine to induce the host immune response, ultimately achieving the goal of controlling tumor growth and distal metastasis. Significant efforts have been devoted to enhancing the immune response caused by phototherapy-eliminated tumors. Recently, supramolecular PIT nanoagents based on precise peptide self-assembly design have been employed to improve the efficacy of photoimmunotherapy by utilizing the stability, targeting capability and flexibility of drugs, increasing tumor immunogenicity and realizing the synergistic amplification of immune effects through multiple pathways and collaborative strategy. This review summarizes peptide-based supramolecular PIT nanoagents for phototherapy-synergized cancer immunotherapy and its progress in enhancing the effect of photoimmunotherapy, especially focusing on the design of peptide-based PIT nanoagents, the progress of bioactive peptides combined photoimmunotherapy, and the synergistic immune-response mechanism.

Potential Role of Traditional Chinese Medicines by Wnt/ß-Catenin Pathway Compared With Targeted Small Molecules in Colorectal Cancer Therapy.

Colorectal cancer (CRC) has become a global public health problem because of its high incidence and mortality rate worldwide. The previous clinical treatment for CRC mainly involves conventional surgery, chemotherapy, and radiotherapy. With the development of tumor molecular targeted therapy, small molecule inhibitors present a great advantage in improving the survival of patients with advanced CRC. However, various side effects and drug resistance induced by chemotherapy are still the major obstacles to improve the clinical benefit. Thus, it is crucial to find new and alternative drugs for CRC treatment. Traditional Chinese medicines (TCMs) have been proved to have low toxicity and multi-target characteristics. In the last few decades, an increasing number of studies have demonstrated that TCMs exhibit strong anticancer effects in both experimental and clinical models and may serve as alternative chemotherapy agents for CRC treatment. Notably, Wnt/ß-catenin signaling pathway plays a vital role in the initiation and progression of CRC by modulating the stability of ß-catenin in the cytoplasm. Targeting Wnt/ß-catenin pathway is a novel direction for developing therapies for CRC. In this review, we outlined the anti-tumor effects of small molecular inhibitors on CRC through Wnt/ß-catenin pathway. More importantly, we focused on the potential role of TCMs against tumors by targeting Wnt/ß-catenin signaling at different stages of CRC, including precancerous lesions, early stage of CRC and advanced CRC. Furthermore, we also discussed perspectives to develop potential new drugs from TCMs via Wnt/ß-catenin pathway for the treatment of CRC.

Cytochrome P450 Mediated Bioactivation of Rutaevin, a Bioactive and Potentially Hepatotoxic Component of Evodia Rutaecarpa.

Rutaevin is one of the major bioactive constituents isolated from Evodia rutaecarpa, a well-known herbal medicine that has been widely prescribed for the treatment of gastrointestinal disorders in China. However, oral administration of rutaevin has been shown to cause hepatotoxicity in mice. Bioactivation was suggested to be involved in rutaevin-induced hepatotoxicity. The aim of this study was to investigate the bioactivation of rutaevin in rat and human liver microsomes fortified with NADPH. Rutaevin was metabolized into the reactive intermediate cis-butene-1,4-dial (BDA) that was dependent on NADPH. The rutaevin-derived BDA intermediate was trapped by nucleophiles such as glutathione (GSH), N-acetyl-lysine (NAL), and methoxylamine (MOA) in the microsomal incubation system. A total of 10 conjugates resulting from the conjugation of the intermediate with GSH, NAL, or MOA were detected and structurally characterized by liquid chromatography combined with high-resolution tandem mass spectrometry. M1, structurally confirmed by NMR spectroscopic analysis, was identified as a cyclic mono(GSH) conjugate of the BDA intermediate, which was also found in the biliary samples of rutaevin-treated rats. Further inhibitory experiments suggested that ketoconazole showed strong inhibitory effect on the formation of the rutaevin-derived BDA intermediate. CYP3A4 was demonstrated to be the major enzyme responsible for rutaevin bioactivation by using cDNA-expressed human recombinant cytochrome P450 enzymes. Additionally, it was found that rutaevin was a mechanism-based inactivator of CYP3A4, with inactivation parameters of KI = 15.98 µM, kinact = 0.032 min-1, and t1/2 inact = 21.65 min. In summary, these findings are of great significance in understanding the bioactivation mechanism of rutaevin, the potential mechanism of rutaevin-caused hepatotoxicity, and the drug-drug interactions associated with rutaevin mainly via CYP3A4 inactivation.

Self-assembled injectable biomolecular hydrogels towards phototherapy.

Biomolecular hydrogels assembled from biomolecules, such as proteins, peptides, and polysaccharides, are promising candidates for facilitating biomedical applications due to their advantages of high biocompatibility, adjustable mechanical properties, functional diversity, and good degradability. This review focuses on current progress in the field of supramolecular injectable biomolecular hydrogels and their applications in antitumor photodynamic therapy (PDT), photothermal therapy (PTT), combined PDT and PTT, and antibacterial phototherapy with emphasis on biomolecular hydrogelators, injectable behaviors, phototherapeutic functions, and the remaining challenges. We hope that this review can provide useful inspiration for the construction and biological applications of novel photo-functional hydrogels as well as phototherapies.

Increased Expression of Kv10.2 in the Hippocampus Attenuates Valproic Acid-Induced Autism-Like Behaviors in Rats.

The role of potassium channels provides suggestive evidence for the etiology of autism. The voltage-gated potassium channel Kv10.2 (KCNH5) is widely expressed in the brain. However, the inherent relationship between Kv10.2 and autism is still unclear. Herein, a rat valproic acid (VPA)-induced autism spectrum disorder model was established. The expression level of Kv10.2 was obviously decreased in the hippocampus of VPA rats. Kv10.2 was mainly localized in neurons. Subsequently, a recombinant lentivirus expressing Kv10.2 was used to upregulate the expression of Kv10.2 in the hippocampus of VPA-exposed rats. The results were promising as injection of the Kv10.2 lentivirus in the hippocampus relieved anxiety and stereotypical behavior, and improved the social and exploratory abilities of rats that were prenatally exposed to VPA. In addition, spectral analysis of electroencephalogram data revealed that animals exposed to VPA exhibited increased high-frequency activity compared with the control rats, and this activity recovered to a certain extent after upregulation of Kv10.2 expression by lentivirus injection. These results suggest that changes in Kv10.2 may play an important role in the etiology of autism, thus providing a promising direction for further research on autism.

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling.

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil (CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain (MLC) phosphorylation. Meanwhile, the siRNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation, suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C. tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

MiR-539-5p negatively regulates migration of rMSCs induced by Bushen Huoxue decoction through targeting Wnt5a.

Bone fractures are very common, and above 5% of the fractures are impaired, leading to nonunions and severe disablilities. The traditional Chinese medicine Bushen Huoxue decoction (BHD) has been used to treat fracture in China. Our previous report has found that BHD promotes migration of rat mesenchymal stem cells (rMSCs) by activating Wnt5a signaling pathway. However, whether and how miRNAs are involved in modulating rMSCs migration induced by BHD has not been explored. In the present study, miRNA microarray analysis and further validation by real-time quantitative RT-PCR revealed that miR-539-5p was down-regulated in BHD-induced rMSCs. Transfection of miR-539-5p mimics suppressed rMSCs migration while the miR-539-5p inhibitor promoted rMSCs migration. Our results suggested that miR-539-5p was a negative regulator of migration of rMSCs induced by BHD. Target prediction analysis tools and Dual-luciferase reporter gene assay identified Wnt5a as a direct target of miR-539-5p. MiR-539-5p inhibited the expression of the Wnt5a and its downstream signaling molecules including JNK, PKC and CaMKII, which played a critical role in regulating migration of rMSCs. Taken together, our results demonstrate that miR-539-5p negatively regulates migration of rMSCs induced by BHD through targeting Wnt5a. These findings provide evidence that miR-539-5p should be considered as an important candidate target for the development of preventive or therapeutic approaches against bone nonunions.

Huo Xue Tong Luo capsule ameliorates osteonecrosis of femoral head through inhibiting lncRNA-Miat.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine has a long history of treating various bone diseases including osteoporosis and osteonecrosis etc. In clinical treatment, Huo Xue Tong Luo capsule (HXTL capsule) containing Peach kernel, Safflower carthamus, Angelica sinensis, Ligusticum wallichii etc, is one of the mostly used prescriptions for treating osteonecrosis of the femoral head (ONFH) with promising effects. OBJECTIVES: This study aims to identify the underlying molecular mechanism of how HXTL capsule exerts its function to ameliorate ONFH. MATERIALS AND METHODS: All femoral bone tissues were collected during surgeries. Rat bone marrow mesenchymal stem cells (rMSCs) were used. Quantitative real time PCR was used to check the relative expression levels of genes. ChIP assay was performed to evaluate the binding of H3K4me3 and H3K27me3 in Miat promoter. RESULTS: We showed that HXTL capsule promoted osteogenesis in rat MSCs as demonstrated by quantitative real time PCR and Alizarin Red S staining. Then we found silencing the endogenous lncRNA-Miat could promote osteogenesis of rMSCs. In addition, the ChIP assay showed that HXTL capsule significantly increased occupancy of H3K27me3 and decreased H3K4me3 in promoter regions of Miat, meaning HXTL capsule inhibited Miat expression through histone modifications. At last, by examining the femoral heads samples obtained from patients with ONFH during total hip arthroplasty surgery, we found the RNA level of hMiat in necrotic tissue was much higher than that of normal tissue. CONCLUSIONS: Taken together, our study shows that lncRNA-Miat might play an important role in pathogenesis of ONFH, and HXTL capsule can promote osteogenesis to ameliorate ONFH through inhibiting the transcriptional expression of Miat, at least partially.

Covalent Assembly of Amphiphilic Bola-Amino Acids into Robust and Biodegradable Nanoparticles for In Vitro Photothermal Therapy.

Organic photothermal sensitizers, such as indocyanine green (ICG), have been widely explored in photothermal therapy as a good substitute for inorganic materials owing to their advantageous biosafety and strong absorption in the near-infrared region. However, their intrinsic low stability and rapid clearance from the body requires further modification for efficient therapeutic application. In this work, we employed a covalent assembly strategy by covalently cross-linking genipin and a functional bola-amino acid to fabricate stable and degradable nanoparticles capable of loading ICG. The covalent assembly introduced strong covalent interactions in the assembly system together with functional linkers, which led to both enhanced stability and extended functionalities. This is distinguished from the conventional supramolecular strategy that relies only on weak noncovalent interactions. The functional building unit, consisting of phenylalanine and the disulfide bond, enables both good assembly and controllable degradation owing to the disulfide bond that responds to glutathione. The assembled nanoparticles show high stability, negligible toxicity, and considerable biodegradability. After loading ICG, the ICG-loaded nanoparticles possessed high photothermal conversion efficiency, and showed an enhanced photothermal effect in the near-infrared region. This covalent assembly strategy could be extended to various biomolecules containing a primary amino group for the fabrication of efficient and multifunctional nanomaterials used in biomedical applications.

[Summarize drug dosage forms in treatment of age-related macular degeneration disease].

In this review, the authors summarized the drugs in treatment of the age-related macular degeneration (AMD or ARMD), including the pathogenesis of the age-related macular degeneration at home and abroad, dosage forms used in the treatment, and the drugs research and development directions in the future. AMD disease is the third largest blinding diseases all over the world, with an incidence of 6.62%. The dosage form of the traditional medicine is mostly oral formulations, playing a role in body, while the newly dosage form is topical drug delivery formulation. Traditional Chinese medicine (TCM) has certain advantages in the treatment of AMD disease and the development of topical drug delivery preparations with newly preparation technologies would have a very bright prospect in the future.

Role of the p-Coumaroyl Moiety in the Antioxidant and Cytoprotective Effects of Flavonoid Glycosides: Comparison of Astragalin and Tiliroside.

The aim of this study was to explore the role of p-coumaroyl in the antioxidant and cytoprotective effects of flavonoid glycosides. The antioxidant effects of astragalin and tiliroside were compared using ferric ion reducing antioxidant power, DPPH• scavenging, ABTS•⁺ scavenging, •O2- scavenging, and Fe2+-chelating assays. The results of these assays revealed that astragalin and tiliroside both exhibited dose-dependent activities; however, tiliroside exhibited lower IC50 values than astragalin. In the Fe2+-chelating assay, tiliroside gave a larger shoulder-peak at 510 nm than astragalin, and was also found to be darker in color. Both of these compounds were subsequently evaluated in a Fenton-induced mesenchymal stem cell (MSC) damaged assay, where tiliroside performed more effectively as a cytoprotective agent than astragalin. Tiliroside bearing a 6''-O-p-coumaroyl moiety exhibits higher antioxidant and cytoprotective effects than astragalin. The 6''-O-p-coumaroyl moiety of tiliroside not only enhances the possibility of electron-transfer and hydrogen-atom-transfer-based multi-pathways, but also enhances the likelihood of Fe-chelating. The p-coumaroylation of the 6"-OH position could therefore be regarded as a potential approach for improving the antioxidant and cytoprotective effects of flavonoid glycosides in MSC implantation therapy.

(+)-Cholesten-3-one induces osteogenic differentiation of bone marrow mesenchymal stem cells by activating vitamin D receptor.

In our previous reports, it was revealed that steroids in traditional Chinese medicine (TCM) have the therapeutic potential to treat bone disease. In the present study, an in vitro model of a vitamin D receptor response element (VDRE) reporter gene assay in mesenchymal stem cells (MSCs) was used to identify steroids that enhanced osteogenic differentiation of MSCs. (+)-cholesten-3-one (CN), which possesses a ketone group that is modified in cholesterol and cholesterol myristate, effectively promoted the activity of the VDRE promoter. Phenotypic cellular analysis indicated that CN induced differentiation of MSCs into osteogenic cells and increased expression of specific osteogenesis markers, including alkaline phosphatase, collagen II and Runt-related transcription factor 2. Furthermore, CN significantly increased the expression of osteopontin, the target of the vitamin D receptor (VDR), which indicated that CN may activate vitamin D receptor signaling. Over-expression of VDR or knockdown studies with VDR-small interfering RNA revealed that the pro-differentiation effects induced by CN required VDR. Furthermore, the present study determined that the C-terminal region of the VDR is responsible for the action of CN. Taken together, the present findings demonstrated that CN induced osteogenic differentiation of MSCs by activating VDR. The present study explored the regulation of stem cells by using a series of similar steroids and provided evidence to support a potential strategy for the screening of novel drugs to treat bone disease in the future.

Anti-inflammatory effect of the blueberry anthocyanins malvidin-3-glucoside and malvidin-3-galactoside in endothelial cells.

Blueberry fruits have a wide range of health benefits because of their abundant anthocyanins, which are natural antioxidants. The purpose of this study was to investigate the inhibitory effect of blueberry's two main anthocyanins (malvidin-3-glucoside and malvidin-3-galactoside) on inflammatory response in endothelial cells. These two malvidin glycosides could inhibit tumor necrosis factor-alpha (TNF-α) induced increases of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) production both in the protein and mRNA levels in a concentration-dependent manner. Mv-3-glc at the concentration of 1 µM could inhibit 35.9% increased MCP-1, 54.4% ICAM-1, and 44.7% VCAM-1 protein in supernatant, as well as 9.88% MCP-1 and 48.6% ICAM-1 mRNA expression (p<0.05). In addition, they could decrease IκBα degradation (Mv-3-glc, Mv-3-gal, and their mixture at the concentration of 50 µM had the inhibition rate of 84.8%, 75.3%, and 43.2%, respectively, p<0.01) and block the nuclear translocation of p65, which suggested their anti-inflammation mechanism was mediated by the nuclear factor-kappa B (NF-κB) pathway. In general malvidin-3-glucoside had better anti-inflammatory effect than malvidin-3-galactoside. These results indicated that blueberry is good resource of anti-inflammatory anthocyanins, which can be promising molecules for the development of nutraceuticals to prevent chronic inflammation in many diseases.

[Effect and mechanism of gastrodin in relaxing isolated thoracic aorta rings in rats].

OBJECTIVE: To investigate the effect of gastrodin in relaxing isolated thoracic aorta rings in rats and discuss its possible mechanism. METHOD: Isotonic tension of isolated thoracic aortic rings in rats with norepineprine (NE) and KCl was recorded to observe the vasodilatory effect of gastrodin and the influence of various drugs on it. RESULT: Gastrodin had the effect in relaxing thoracic aortas with or without endothelium, and there was no significant difference. NG-nitro-L-argininemethylester (L-NAME, 1 x 10(-4) mol x L(-1)), methylene blue (MB, 1 x 10(-5) mol x L(-1)), indomethacin (INDO, 1 x 10(-5) mol x L(-1)) had no effect on the vasodilation action of gastrodin on thoracic aortas precontracted by NE. 4-aminopyrimide (4-AP, 1 x 10(-4) mol x L(-1)), tetrathylamonium (TEA, 1 x 10(-3) mol x L(-1)), BaCl2 (1 x 10(-4) mol x L(-1)) and glibenclamide (Gli, 1 x 10(-5) mol x L(-1)) could inhibit gastrodin's effect in relaxing thoracic aorta rings. In the absence of Ca2+, pre-incubated gastrodin showed a notable inhibitory effect in relaxing NE contraction. CONCLUSION: Gastrodin shows a dose-dependent and endothelium-independent effect in relaxing rat isolated thoracic aorta rings. The mechanism is related to K+ channel, inhibition of release of Ca+ stored in endoplasmic reticulum of vascular smooth muscle cells and inflow of external calcium Ca2+.

[Effect of shugan jianpi bushen recipe on splenic T lymphocytes and virus load in hepatitis B virus transgenic mice].

OBJECTIVE: To observe the effect of Shugan Jianpi Bushen Recipe (SJBR) on the splenic T lymphocytes and virus load in the hepatitis B virus (HBV) transgenic (Tg) mice, and to study its antiviral efficacy and mechanisms of action. METHODS: Sixty male BALB/C mice of SPF grade were included. Ten non-HBV Tg male mice were included as the normal control group. Fifty HBV Tg mice were randomly divided into five groups, i. e., the model group, the adefovir (ADV) group, the low dose SJBR group, the middle dose SJBR group, and the high dose SJBR group, ten in each. 10, 20, and 40 g/kg SJBR crude drug was respectively given by gastro-gavage to mice in the low dose SJBR group, the middle dose SJBR group, and the high dose SJBR group. ADV 50 mg/kg body weight was given by gastrogavage to mice in the ADV group. Equal volume of sterilized iso-osmia was given to mice in the normal group and the model group. The medication was performed once daily, totally for 21 successive days. The serum HBV DNA titers of HBV Tg mice were detected using Real-time fluorescent PCR one day before administration (T0), ten days after administration (T1), 21 days after administration (T2), and three days after withdrawal (T3), respectively; the serum hepatitis B surface antigen (HBsAg) of HBV Tg mice on T3 was detected by ELISA. The splenic T lymphocyte percent of all mice was detected by flow cytometry. RESULTS: Serum HBsAg at TO was positive in the high-, middle-, low-dose SJBR, and ADV groups. The HBsAg negative rate at T3 was lower in the high dose SJBR group than in the ADV group, showing statistical difference (P<0.01). Compared with TO of the same group, the serum HBV DNA titers could be continually decreased by high dose SJBR, showing statistical difference (P<0.01). The serum HBV DNA titers also gradually decreased in the ADV group (P<0.01), but it somewhat increased at T3. The CD3+ cell percent could be elevated by high-, middle-, low-dose SJBR, and ADV groups (P<0.05, P<0.01). The CD8+ T cell percent could also be obviously lowered by high-, middle-, and low-dose SJBR (P<0.01). Compared with the middle-, low-dose SJBR, and ADV groups, the CD4+ T cell percent and CD4+/CD8+ increased as well as CD8+ decreased in the high dose SJBR group, showing statistical difference (P<0.01). The CD3+ T cell percent was significantly positively correlated to the decrement of HBV DNA titers between the pre-treatment and post-treatment in the middle dose SJBR group (r=0.654, P<0.05). The percents of CD4+, CD8+ T cells and CD4+/CD8+ were significantly positively correlated to the decrement of HBV DNA titers between the pre-treatment and post-treatment in the high dose SJBR group (r=0.53, r=0.79, r =0.80, P<0.01). CONCLUSIONS: SJBR were capable of inhibiting the HBV DNA duplication of HBV Tg mice. One of its anti-HBV mechanisms possibly be improving the the abnormality of T lymphocyte subsets and the immune function.

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.

BACKGROUND: Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. RESULTS: Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. CONCLUSIONS: The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.