The effects of protein-rich extract from Rhizoma Gastrodiae against cerebral ischemia/reperfusion injury via regulating MAPK and PI3K/AKT signaling pathway.

BACKGROUND: Rhizoma Gastrodiae is a highly valuable traditional Chinese medicine and functional health food that has been used in China to treat neurological disorders for thousands of years. Rhizoma Gastrodiae contains various of biological activities, such as antioxidative, neuroprotective, learning improvement, anxiolytic, and antidepressant effects. However, no studies have been conducted to explore the effects of the protein components in Rhizoma Gastrodiae (GEPS) and its potential protective effects against ischemic stroke.Our main goal was to investigate the effects of GEPS on ischemia/reperfusion (I/R) injury and its possible mechanisms. METHODS: A middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia mouse model and an oxygen-glucose deprivation (OGD/R) injury model in HT22 cells were established. A neurobehavioral test was performed 24 h after MCAO, and brain infarction was measured. A Morris water maze experiment was conducted on Day 14 after reperfusion in mice. Hematoxylin and eosin (HE) and TUNEL staining were performed to assess apoptotic neuronal death. Immunohistochemical analysis was used to detect BDNF and GAP43 expression. The content of SOD, MDA, GSH-PX and ROS were detected. The protein expression was analyzed using Western blotting. Cell viability was determined by MTT assay. Cell apoptosis was examined by flow cytometry. RESULTS: GEPS reduced apoptosis, decreased cerebral infarction, improved neurological defects, and ameliorated oxidative stress in the ischemic penumbra. In addition, GEPS increased the expression of BDNF and GA43 in the penumbra. Mechanistically, GEPS counteracted MCAO-induced PI3K/AKT inhibition and activation of MAPK signaling pathways. CONCLUSION: GEPS has a clear neuroprotective effect on I/R injury, and its mechanism may be linked to the PI3K/AKT and MAPK signaling pathways.

Transcriptomics reveals the molecular regulation of Chinese medicine formula on improving bone quality in broiler.

Skeletal disorder is of concern to the poultry industry as it affects animal welfare and production performance. Traditional Chinese medicine could improve bone quality and reduce the incidence of bone disease, but the molecular regulation of Chinese medicine formula (CMF) on improving bone quality in broilers is still unclear. This study was performed to research the effects of CMF on skeletal performance of Cobb broilers and reveal the molecular regulation. A total of 120 one-day-old Cobb broilers were randomly allocated into 4 equal groups of 30 chickens, with 5 replicates and 6 chickens in each replicate. The control (CON) group was fed a diet without CMF, while the CMF1, CMF2, and CMF3 groups were supplemented with different CMF at 6,000 mg/kg diet, respectively. The broilers were raised to 60 d of age, then bone tissues were collected for biomechanical properties, micro-CT detection and transcriptomic sequencing analysis. The results showed that CMF3 improved the biomechanical properties of broiler tibia, via increasing the elastic modulus (P < 0.05), yield strength (P > 0.05), maximum stress (P < 0.05) and fracture stress (P < 0.05) of the tibia. Micro-CT analysis indicated that CMF3 increased the bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface density (BS/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and decreased the trabecular separation (Tb.Sp) of femur cancellous bone (P < 0.05). RNA-seq analysis revealed 2,177 differentially expressed genes (DEGs) (|log2FoldChange| ≥ 1, FDR < 0.05) between the CMF3 group and CON group. Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis showed 13 pathways mostly associated with bone growth and development and bone metabolism, and we identified 39 bone-related DEGs. This study suggests that CMF3 could improve bone strength and bone microstructure of broilers, and showed a positive effect on bone performance. Our research could provide a theoretical reference for the development of pollution-free feed additives to improve the skeletal performance of broilers, which could help promote healthy farming of chickens.

Ethyl acetate extract of Gastrodia elata protects Caenorhabditis elegans from oxidative stress and amyloid ß peptide toxicity.

Gastrodia elata Blume is a traditional Chinese medicine with a long history, which has numerous pharmacological activities, such as anti-inflammation, anti-oxidation and protection of nerves. The present study investigated the regulatory effect of ethyl acetate extract of Gastrodia elata (EEGE) on the ß-amyloid (Aß) toxicity of Caenorhabditis elegans (C. elegans). First, the main components of EEGE were analyzed using high-performance liquid chromatography, and the total phenols, total flavonoids and total antioxidant capacity of EEGE were determined. Next, the regulation effect of EEGE on Aß-induced toxicity of C. elegans was evaluated through experiments on nematode paralysis, lifespan, oxidative and heat stress, locomotor ability, reproductive ability, reactive oxygen species (ROS) level, Aß aggregation test, malondialdehyde (MDA) level, catalase (CAT) activity and superoxide dismutase (SOD) activity. Finally, the mechanism of EEGE was elucidated using RNA sequencing (RNA-Seq) and the expression levels of related genes were verified using quantitative PCR. The present study revealed that the main components of EEGE included phosphorylated (p)-hydroxybenzyl alcohol, p-hydroxybenzaldehyde and 4,4'-dihydroxydiphenylmethane, possessing strong in vitro free radical scavenging and reducing abilities. In addition, after the intervention of EEGE, the paralysis of nematodes could be delayed, the survival time of the nematodes was prolonged, the survival rate of the nematodes under stress (high temperature and oxidation) conditions was improved, the activity capacity and reproductive capacity of the nematodes were improved, the activities of SOD and CAT were improved and the levels of ROS and MDA were reduced. Notably, EEGE directly inhibited Aß plaque aggregation in nematodes. RNA-Seq analysis showed that EEGE regulated metabolism and longevity-related genes, and these genes were regulated by the insulin/IGF-1 signaling (IIS) pathway. Therefore, the present study hypothesized that the regulatory mechanism of EEGE was significantly related to the IIS pathway. The present research results demonstrated that the protective effect of EEGE on transgenic C. elegans was to reduce Aß protein aggregation, improve the in vivo antioxidant level, effectively remove free radicals and to regulate the expression of genes related to IIS pathway, thereby reducing Aß-induced toxicity and delaying nematode paralysis.

[Quality control mechanism of mitochondria by 3,4-dihydroxybenzaldehyde through OGT-PINK1 pathway].

Based on the O-GlcNAc transferase(OGT)-PTEN-induced putative kinase 1(PINK1) pathway, the mechanism of 3,4-dihydroxybenzaldehyde(DBD) on mitochondrial quality control was investigated. Middle cerebral artery occlusion/reperfusion(MCAO/R) rats were established. SD rats were randomized into sham operation group(sham), model group(MCAO/R), DBD-L group(5 mg·kg~(-1)), and DBD-H group(10 mg·kg~(-1)). After 7 days of administration(ig), MCAO/R was induced in rats except the sham group with the suture method. Twenty-four h after reperfusion, the neurological function and the percentage of cerebral infarct area were measured. Based on hematoxylin and eosin(HE) staining and Nissl staining, the pathological damage of cerebral neurons was examined. Then the ultrastructure of mitochondria was observed under the electron microscope, and the co-localization of light chain-3(LC3), sequestosome-1(SQSTM1/P62), and Beclin1 was further detected by immunofluorescence staining. It has been reported that the quality of mitochondria can be ensured by inducing mitochondrial autophagy through the OGT-PINK1 pathway. Therefore, Western blot was employed to detect the expression of OGT, mitophagy-related proteins PINK1 and E3 ubiquitin ligase(Parkin), and mitochondrial kinetic proteins dynamin-like protein 1(Drp1) and optic atrophy 1(Opa1). The results showed that MCAO/R group had neurological dysfunction, large cerebral infarct area(P<0.01), damaged morphological structure of neurons, decreased number of Nissl bodies, mitochondrial swelling, disappearance of mitochondrial cristae, decrease of cells with LC3 and Beclin1, rise of cells with P62(P<0.01), inhibited expression of OGT, PINK1, and Parkin, up-regulated expression of Drp1, and down-regulated expression of Opa1 compared with the sham group(P<0.01). However, DBD improved the behavioral deficits and mitochondrial health of MCAO/R rats, as manifested by the improved morphology and structure of neurons and mitochondria and the increased Nissl bodies. Moreover, DBD increased cells with LC3 and Beclin1 and decreased cells with P62(P<0.01). In addition, DBD promoted the expression of OGT, PINK1, Parkin, and Opa1 and inhibited the expression of Drp1, enhancing mitophagy(P<0.05, P<0.01). In conclusion, DBD can trigger PINK1/Parkin-mediated brain mitophagy through the OGT-PINK1 pathway, which plays a positive role in maintaining the health of the mitochondrial network. This may be a mitochondrial therapeutic mechanism to promote nerve cell survival and improve cerebral ischemia/reperfusion injury.

Network pharmacology and molecular docking analysis on molecular targets and mechanisms of Gastrodia elata Blume in the treatment of ischemic stroke.

Gastrodia elata Blume (GEB) is widely used to treat cardio-cerebrovascular disease in China and in traditional Chinese medicine it is considered to be a dispelling wind and dredging collateral. However, the mechanism and active components of the plant in treating ischemic stroke (IS) remain unclear. The present study aimed to identify the active components and mechanism of GEB in treating IS using network pharmacology and molecular docking technology. Network analysis predicted 752 potential targets from 14 compounds in GEB, sharing 32 key targets with IS-associated targets. Gene Ontology analysis of key targets showed that 'oxidative stress', 'immune response' and 'regulation of blood circulation' were significantly enriched. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the key targets regulated 11 representative pathways including 'arachidonic acid metabolism', 'lipid and galactose metabolism'. In the protein-protein interaction network, five core targets, including toll-like receptor agonist, STAT3, myeloperoxidase (MPO), prostaglandin-endoperoxide synthase and matrix metalloproteinase (MMP)9, were identified and successfully docked with four active components: Palmitic acid, alexandrin, para-hydroxybenzaldehyde and gastrodin. Alexandrin, para-hydroxybenzaldehyde, and gastrodin are closely related to brain ischemia/reperfusion damage and repair. Therefore, to further verify the mechanism of action of three active components in the second part, we established the HT22 oxygen-glucose deprivation-reperfusion (OGD/R) model. Cell Counting Kit-8 assay and western blot analysis demonstrated that these three active components of GEB regulated core targets of molecular docking, such as STAT3, MPO and MMP9. In vitro experiments showed that OGD/R decreased cell survival, while this effect was reversed by the three active components of GEB. In addition, western blot analysis indicated that alexandrin upregulated expression of phosphorylated-STAT3, para-hydroxybenzaldehyde downregulated MPO and gastrodin downregulated MMP9. Therefore, the present study showed that GEB may prevent and treat IS via interaction between the active components and the main targets, which is key for investigating the efficacy of traditional Chinese medicine.

[Effect of ethanol extract of Gastrodiae Rhizoma on mitochondrial dysfunction in cerebral ischemia-reperfusion injury].

This study observed the effects of ethanol extract of Gastrodiae Rhizoma(GE) on multiple aspects of mitochondrial dysfunction by investigating the mitochondria isolated from rat brains subjected to focal middle cerebral artery occlusion/reperfusion(MCAO/R). SD rats were randomly divided into a sham operation group(Sham), a model group(MCAO/R), low-and high-dose ethanol extract of GE groups(262.3 and 524.6 mg·kg~(-1)), and a nimodipine group(Nim, 15 mg·kg~(-1)). After continuous intragastric administration for 7 days, the MCAO/R model was induced in rats by the suture method. The neurological function and percentage of cerebral infarction volume were measured 24 h after reperfusion, and mitochondrial ultrastructure was observed under an electron microscope. Mitochondria were separated by gradient centrifugation and detected for reactive oxygen species(ROS), malondialdehyde(MDA), respiratory chain enzyme complex Ⅰ-Ⅳ activity, mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), and mitochondrial adenosine triphosphate(ATP) content. Enzyme-linked immunosorbent assay(ELISA) was used to detect the expression of cytochrome C(Cyt C) in different sites. TUNEL staining was used to observe the apoptosis of brain tissues in each group, and Western blot was used to detect the expression of B-cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax) in brain tissues. The experimental results revealed that compared with the Sham group, the MCAO/R group showed evident neurological dysfunction and cerebral infarction(P<0.01) accompanied by mitochondrial swelling and crest disappearance, increased ROS level and MDA content, inhibited activity of respiratory chain enzyme complex Ⅰ-Ⅳ, abnormal opening of mPTP, and reduced MMP and mitochondrial ATP(P<0.01). Besides, many Cyt C was released from mitochondria into the cytoplasm to induce apoptosis(P<0.01). The ethanol extract of GE positively affected the behavior deficit and mitochondrial health of MCAO/R rats, with the manifestations of decreased production of ROS and MDA(P<0.01), potentiated activity of mitochondrial respiratory chain enzyme complex Ⅰ-Ⅳ, and restored the level of mPTP(P<0.05). In addition, the ethanol extract of GE reduced the loss of MMP and the escape of Cyt C to the cytoplasm(P<0.05), reduced the number of TUNEL positive cells(P<0.01) accompanied by the decrease in Bax and the up-regulation of Bcl-2(P<0.01), and increased the level of ATP(P<0.01). In conclusion, GE ethanol extract has a protective effect against MCAO/R-induced mitochondrial dysfunction, and the mechanism may be related to the regulation of oxidative stress, maintenance of functional morphology of mitochondria, and inhibition of endogenous apoptosis.

Inflammatory Cytokines, Adipocytokines, and Th17/Treg Balance in Patients with Nonalcoholic Fatty Liver Disease following Administration of Dahuang Zhechong Pills.

Objectives: The occurrence and development of nonalcoholic fatty liver disease (NAFLD) is related to lipid peroxidation, imbalance of inflammatory response factors, and immune function disorder. This study was conducted with the purpose of investigating the expression levels of inflammatory cytokines and adipocytokines and Th17/Treg balance in NAFLD patients treated with Dahuang Zhechong pills (DHZCPs). Methods: The study recruited 100 NAFLD patients who were then arranged into the test group and control group. Patients in the test group were treated with DHZCPs, while patients in the control group were untreated. Peripheral TH17 and Treg cells were detected by flow cytometry, and peripheral IL-17, IL-10, hs-CRP, and TNF-α expression levels were determined by enzyme-linked immunosorbent assay (ELISA) methods. The concentrations of ghrelin, leptin, and adiponectin were quantitatively examined. Results: The levels of TC, TG, ALT, and AST were declined but the level of HDL-C was increased in NAFLD patients treated with DHZCPs compared with untreated patients (P < 0.05). The ratio of Th17/Treg in NAFLD patients treated with DHZCPs was (1.52 ± 0.21), which was significantly lower than (2.39 ± 0.45) of untreated patients (P < 0.05). The levels of IL-17, hs-CRP, and TNF-α were lower, but the level of IL-10 was higher in NAFLD patients treated with DHZCPs than that in untreated patients (P < 0.05). The expression levels of ghrelin and adiponectin in NAFLD patients treated with DHZCPs were evidently higher than those in untreated patients (P < 0.01), and the expression level of leptin in NAFLD patients treated with DHZCPs was evidently lower than that in untreated patients (P < 0.01). Conclusions: Administration of DHZCPs regulates the immune function of NAFLD patients by keeping Th17/Treg balance and affecting the levels of inflammatory cytokines and adipocytokines.

[Effect of Desmodium renifolium on ovariectomized rat model of osteoporosis via BMP-2/Smads signaling pathway].

This study aimed to investigate the effect of Desmodium renifolium(Linn.) Schindl on ovariectomized rat model of osteoporosis and explore the underlying mechanism. Rats were randomized into a sham group, a model group, a Xianlin Gubao group, and low-, medium-, and high-dose D. renifolium groups. Bilateral oophorectomy was performed in other groups except sham group(removal of bilateral peri-ovarian adipose tissue). The sham group and model group were administrated with equal volume of 0.5% CMC-Na, Xianlin Gubao group with Xianlin Gubao, and D. renifolium groups with different concentrations of alcoholic extract of D. renifolium once a day for 14 weeks. The body weight of rats were recorded during the experiment. The levels of estradiol(E_2), 1,25-dihydroxyvitamin D_3 [1,25(OH)_2D_3], calcium(Ca), and phosphorus(P) in serum were determined after the administration. The femur microstructure was analyzed via micro-CT. RT-PCR and Western blot were employed to respectively determine the mRNA and protein levels of bone morphogenetic protein 2(BMP-2), Runt-related transcription factor 2(Runx2), and osterix(OSX) in the tibia of rats. The results indicated that D. renifolium significantly inhibited the weight growth, improved the uterus appearance, elevated the levels of E_2, Ca, P, and 1,25(OH)_2D_3 in serum, increased the number and reduced the fracture of bone trabeculae, ameliorated the bone microstructure parameters, and up-regulated the mRNA and protein levels of BMP-2, Runx2, and OSX. All the results demonstrated that D. renifolium had significant protective effect on the ovariectomized rat model of osteoporosis by regulating the BMP-2/Smads signaling pathway.

Identification of SARS-CoV-2 inhibitors using lung and colonic organoids.

There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.

Antidepressant-like mechanism of honokiol in a rodent model of corticosterone-induced depression.

Depression is closely linked to hypothalamus-pituitary-adrenal axis hyperactivity. Honokiol, a biphenolic lignan compound obtained from the traditional Chinese medicine Magnolia officinalis, can reduce the activity of the hypothalamus-pituitary-adrenal axis and improve depression-like behavior caused by hypothalamus-pituitary-adrenal axis hyperactivity. The current study investigated the specific mechanism of action of this effect. A depression model was established by repeated injections of corticosterone to study the antidepressant-like effect of honokiol and its potential mechanism. Honokiol prevented the elevated activity of the hypothalamus-pituitary-adrenal axis and the depression-like behavior induced by corticosterone. Treatment with honokiol resulted in greater glucocorticoid receptor mRNA expression, greater glucocorticoid receptor-positive expression, and a greater ratio of glucocorticoid receptor to the mineralocorticoid receptor in the hippocampus. Moreover, honokiol treatment led to lower levels of interleukin-1ß in serum and the positive expression of the interleukin-1ß receptor in the hippocampus. These results demonstrate that the antidepressant-like mechanism of honokiol, which has effects on inflammatory factors, may act through restoring the typical activity of the hypothalamus-pituitary-adrenal axis by regulating the glucocorticoid receptor-mediated negative feedback mechanism and the balance between glucocorticoid and mineralocorticoid receptors.

A Natural CCR2 Antagonist Relieves Tumor-associated Macrophage-mediated Immunosuppression to Produce a Therapeutic Effect for Liver Cancer.

Hepatocellular carcinoma (HCC) is a common malignant tumor in the digestive tract with limited therapeutic choices. Although sorafenib, an orally administered multikinase inhibitor, has produced survival benefits for patients with advanced HCC, favorable clinical outcomes are limited due to individual differences and resistance. The application of immunotherapy, a promising approach for HCC is urgently needed. Macrophage infiltration, mediated by the CCL2/CCR2 axis, is a potential immunotherapeutic target. Here, we report that a natural product from Abies georgei, named 747 and related in structure to kaempferol, exhibits sensitivity and selectivity as a CCR2 antagonist. The specificity of 747 on CCR2 was demonstrated via calcium flux, the binding domain of CCR2 was identified in an extracellular loop by chimera binding assay, and in vivo antagonistic activity of 747 was confirmed through a thioglycollate-induced peritonitis model. In animals, 747 elevated the number of CD8+ T cells in tumors via blocking tumor-infiltrating macrophage-mediated immunosuppression and inhibited orthotopic and subcutaneous tumor growth in a CD8+ T cell-dependent manner. Further, 747 enhanced the therapeutic efficacy of low-dose sorafenib without obvious toxicity, through elevating the numbers of intra-tumoral CD8+ T cells and increasing death of tumor cells. Thus, we have discovered a natural CCR2 antagonist and have provided a new perspective on development of this antagonist for treatment of HCC. In mouse models of HCC, 747 enhanced the tumor immunosuppressive microenvironment and potentiated the therapeutic effect of sorafenib, indicating that the combination of an immunomodulator with a chemotherapeutic drug could be a new approach for treating HCC.

Pharmacokinetics and effects of demographic factors on blood 25(OH)D3 levels after a single orally administered high dose of vitamin D3.

AIM: To examine the biological consequences and demographic factors that might affect the pharmacokinetics of vitamin D3 after a single high dose intervention in a young Chinese population with vitamin D insufficiency status. METHODS: A total of 28 young subjects (25 to 35 years old) with vitamin D insufficiency status [serum 25(OH)D <30 ng/mL] was recruited in Shanghai, China. The subjects were orally administered a single high dose of vitamin D3 (300 000 IU). Baseline characteristics and blood samples were collected at d 0, 1, 2, 3, 7, 28, 56, 84 and 112 after the intervention. The blood biomarker levels were determined with standardized methods. RESULTS: The intervention markedly increased the blood 25(OH)D3 levels within the first five days (mean Tmax=5.1±2.1 d) and sustained an optimal circulating level of 25(OH)D3 (≥30 ng/mL) for 56 d. After the intervention, body weight and baseline 25(OH)D3 levels were significantly correlated with circulating 25(OH)D3 levels. No adverse events and no consistently significant changes in serum calcium, creatinine, glucose, parathyroid hormone, vitamin D binding protein, or the urinary calcium/reatinine ratio were observed. However, there was a significant increase in phosphorus after the vitamin D3 intervention. Total cholesterol and triglyceride levels were decreased at the end of the trial. CONCLUSION: The pharmacokinetics of vitamin D after intervention were influenced by baseline 25(OH)D3 levels and the body weight of the subjects. The results suggest that a single high oral vitamin D3 intervention is safe and efficient for improving the vitamin D status of young Chinese people with vitamin D insufficiency.

Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats via inhibiting MMP2/9, TIMP1/2 and the TGF-ß/Smad signaling pathways.

AIM: Chinese medicine CGA formula consists of polysaccharide from Cordyceps sinensis mycelia (CS-PS), gypenosides and amygdalin, which is derived from Fuzheng Huayu (FZHY) capsule for treating liver fibrosis. In this study we attempted to confirm the therapeutic effects of CGA formula in dimethylnitrosamine (DMN)-induced liver fibrosis in rats, and to identify the mechanisms of anti-fibrotic actions. METHODS: Rats were injected with DMN (10 mg·kg(-1)·d(-1), ip) for 3 consecutive days per week over a 4-week period. The rats then were orally administered with CGA formula (CS-PS 60 mg·kg(-1)·d(-1), gypenosides 50 mg·kg(-1)·d(-1) and amygdalin 80 mg·kg(-1)·d(-1)) daily in the next 2 weeks. CS-PS, gypenosides or amygdalin alone were administered as individual component controls, whereas colchicine and FZHY were used as positive controls. Serum biomarkers were measured. Hepatic injury, collagen deposition and stellate cell activation were examined. The MMP activities, expression of TIMP protein and proteins involved in the TGF-ß1/Smad signaling pathways in liver tissues were assayed. RESULTS: In DMN-treated rats, administration of CGA formula significantly decreased serum ALT, AST and total bilirubin and hepatic hydroxyproline levels, increased serum albumin level, and attenuated liver fibrosis as shown by histological examination. Furthermore, these effects were comparable to those caused by administration of FZHY, and superior to those caused by administration of colchicine or the individual components of CGA formula. Moreover, administration of CGA formula significantly decreased the protein levels of α-SMA, TGF-ß1, TGF-ß1 receptor (TßR-I), p-TßR-I, p-TßR-II, p-Smad2, p-Smad3, TIMP1 and TIMP2, as well as MMP2 and MMP9 activities in liver tissues of DMN-treated rats. CONCLUSION: Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats, and this effect was likely associated with the down-regulation of MMP2/9 activities, TIMP1/2 protein expression and the TGF-ß1/Smad signaling pathways in the liver.

PROTECTIVE EFFECTS OF ETHYL ACETATE EXTRACTION FROM GASTRODIA ELATA BLUME ON BLOOD-BRAIN BARRIER IN FOCAL CEREBRAL ISCHEMIA REPERFUSION.

BACKGROUND: Damage of the blood brain barrier (BBB) during the process of cerebral ischemic injury is a key factor which influences the therapeutic efficacy to the cerebral ischemic injury. The present study was designed to verify the mechanisms underlying the protective effects of the ethyl acetate (EtOAc) extraction from Gastrodia elata Blume (GEB) on the BBB by developing a model of cerebral ischemia-reperfusion in rats. MATERIAL AND METHODS: MCAO/R model in rats was developed through a thread embolism method. The neurological scales, the moisture and the evans blue (EB) contents of brains were detected. Meanwhile, the release of nitric oxide (NO) and activities of NO synthase (NOS) in brain tissues were measured. Western blotting analyses were also performed to assess the protein expressions of AQP-4, Occludin and Claudin-5 in brain tissue. RESULTS: After rats were pretreated with different concentrations of EtOAc extractions from GEB, the neurologic scores, the EB contents in the brain tissues and the moisture of the brains were significantly decreased. Meanwhile, the release of NO, the activities of nNOS and iNOS were notably inhibited. Furthemore, the protein expression of AQP-4 was markedly decreased, but the protein expressions of -5 and Occludin were significantly increased. CONCLUSION: the EtOAc extracts of GEB may decrease the permeability of BBB when focal cerebral ischemia occurs. The inhibition of the NOS pathways, the attenuation of the protein expression of AQP-4 and the enhancement of the expressions of the tight junction proteins may contribute to the protective effects of the EtOAc extracts from GEB on BBB.

Neuroprotective effect of ethyl acetate extract from gastrodia elata against transient focal cerebral ischemia in rats induced by middle cerebral artery occlusion.

OBJECTIVE: To investigate the protective effect of neuroprotection against transient focal cerebral ischemia of the extract from Tianma (Rhizoma Gastrodiae) and the possible mechanisms underlying the action. METHODS: Cerebral ischemia-reperfusion injury was induced through middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were randomly divided into four groups: sham-operated, ischemia-reperfusion model, 102.6 mg/kg extract treated and 11.4 mg/kg extract treated groups. The extract was prepared from gastrodia elata with ethyl acetate. The effect of the extract tested on rat neurological deficits and Cerebral index, cerebral infarct volume, brain injury, terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and B-cell lymphoma-2 (Bcl-2) positive cells. RESULTS: The extract was able to reduce neurological scores, cerebral index and cerebral infarction rate. The brain injury was also relieved by the extract. The results of immunofluorescence staining analysis indicated that the extract increased the expression of Bcl-2 and reduced TUNEL-positive cells significantly in the extract treated groups. CONCLUSION: These results suggested that the extract relieved ischemic injury induced by transient focal cerebral ischemia in rats, and this neuroprotective effect might be partially due to the attenuated apoptosis pathway.

[A brief analysis on the on-duty systems and dispatching systems in the Imperial Academy of Medicine of the Qing Dynasty].

The "on-duty systems" and "dispatching systems" in the Tai yi yuan (Imperial Academy of Medicine) of the Qing Dynasty served as the "prelude" of the medical officials to carry out their diagnoses and treatments. Mainly serving in the royal court, the "on-duty systems" included 3 sorts, viz., "specially selected on-duty", "interior on-duty" and "exterior on-duty". In addition to providing medical services in the royal court, Imperial Academy of Medicine also served other royal members in the Capital, the ministers, the military, the civilians, the metropolitan examinations and the prisons. Thus, the "dispatching systems" was established, also included 3 sorts, viz., "special dispatchments", "dispatchments through reporting to the emperor" and "dispatchments through official communication".

[Study on the chemical constituents of Gastrodia elata].

OBJECTIVE: To study the chemical constituents of Gastrodia elata. METHODS: The compounds were isolated by silica gel column chromatography and recrystallization and their structures were elucidated by spectral analysis. RESULTS: The structures of 14 compounds were identified as: p-hydroxybenzyl alcohol (1), p-hydroxybenzaldehyde (2), 3, 4-dihydroxybenzaldehyde (3), 4, 4'-dihydroxy, dibenzyl ether (4), 4-hydroxymethyl benzyl-4'-hydroxy-3'-(4"-hydroxy benzl) benzyl ether (5), 4-ethoxybenzyl alcohol (6), anisic alcohol (7), bis(3, 4-dihydroxyphenyl) methane (8), 4, 4'-dihydroxydiphenyl methane (9), 2, 4-bis(4'-hydroxy-benzyl)-phenol (10), 4-( methoxymenthyl) benzene-1, 2-diol (11), p-methylphenyl-1 -O-beta-D-glucopyranoside (12), N6-(4'-hydroxybenzl) -adenosine (13) and beta-sitosterol (14). CONCLUSION: Compounds 6 - 8 and 11 are isolated from this plant for the first time.

[Effect of gypenosides on DMN-induced liver fibrosis in rats].

OBJECTIVE: To study the effect of gypenosides on DMN-induced liver fibrosis in rats. METHOD: A rat liver fibrosis model was established by injecting DMN intraperitoneally. Four weeks later, model rats were randomly devided into three groups: the model group, the gypenosides treated group (200 mg x kg(-1)) and the colchicine treated group (0.1 mg x kg(-1)), with 10 specimens for each group. After a 2-week treatment, following parameters were observed: (1) last body weight, weight ratio between liver and spleen; (2) content of liver hydroxyproline (Hyp); (3) activity of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (gamma-GT), content of albumin (Alb) and total bilirubin( TBiL) in serum; (4) liver pathology (Sirius red staining and HE staining); (5) activity of liver superoxide dismutase (SOD), glutathione reduced (GSH), glutathione peroxidase (GSH-Px) and content of liver maleic dialdehyde (MDA). RESULT: There were classic liver cirrhosis pathological changes in model groups. Compared with the normal group, liver Hyp content, activity of serum ATL, AST, gamma-GT and content of serum TBiL, MDA of model groups significantly increased; content of serum Alb and liver GSH, activity of liver SOD and GSH-Px decreased significantly in model groups. In comparison with the model group, liver cirrhosis remarkable improved in the gypenosides group, content of liver Hyp reduced significantly (P < 0.01), which was equal to the colchicine group. Compared with the model group, liver function parameters improved markedly in the gypenosides group; liver SOD and GSH-Px activities significantly increased; MDA content reduced significantly (P < 0.05). CONCLUSION: Gypenosides shows an effect in treating DMN-induced liver fibrosis in rats.

[Uniform designed research on the active ingredients assembling of Chinese medicine prescription for anti-liver fibrosis].

OBJECTIVE: To explore the method and significance for studying active anti-liver fibrosis ingredients consisted Chinese medicine compound prescription based on Chinese medicine theory. METHODS: Optimized prescription was screened out, adopting uniform block design with 4-factor 8-level table and regression analysis, through applying the four known effective ingredients (cordyceps sinensis polysaccharide, salvianolic acid B, amygdaloside and gypenosides) of Fuzheng Huayu Capsule (FZHYC, a new Chinese medine anti-liver fibrosis drug) to two rat liver fibrosis models established separately by dimethylnitrosamine (DMN) and CCl4, and taking the liver content of hydroxyproline (Hyp) as the screen index. Then a further study for comparing and verifying the efficacy of the obtained optimized prescription was conducted on the two former models respectively by observing the changes of Hyp content in liver, serum ALT activity and fibrosis pathology after medication, controlled by the original FZHYC and the recipe assembled by all the four ingredients. RESULTS: Two optimized prescriptions (OPA and OPB) were screened out separately in the studies conducted on the two models. Both of them were consisted of cordyceps sinensis polysaccharide, Amygdaloside and Gypenosides, but different in constituent ratio, i.e., the ratio in OPA was 60 : 80: 50, and that in OPB, 20: 160: 50. Verifying study showed both OPA and OPA were significantly effective, with the efficacy equivalent to that of FZHYC (P>0.05). However, when they were used in combining with salvianolic acid B (the cutout ingredient in the screening), the efficacy lowered surely. CONCLUSIONS: Uniform design is a valuable method in the compatibility research of Chinese Medicine drugs' composition. To assemble a new compound recipe reasonably based on the prescription of traditional compound recipe could make its effect equivalent to that of the original prescription. Ingredients or constituents in a prescription, either presented synergistic or antagonistic effects, are not randomly stacked together, and they should be orderly assembled in intrinsic rules of qualitative and quantitative changing.