Animal protein-plant protein composite nanospheres for dual-drug loading and synergistic cancer therapy.

The co-delivery of multiple drugs using one drug carrier is a viable strategy to optimize drug dosage and reduce the side effects in chemotherapy. Herein, a hydrophilic animal protein (silk fibroin) and a hydrophobic plant protein (zein) were selected for preparing a composite drug carrier. Adapting our previously developed method for the preparation of regenerated silk fibroin (RSF) nanospheres, we prepared RSF/zein nanospheres that displayed an interesting structure including a single central hole. The particle size of the RSF/zein nanospheres was regulated from 150 to 460 nm by varying the preparation conditions, implying that such a drug carrier is suitable for both intravenous administration and lymphatic chemotherapy. Two anti-cancer drugs with different target sites, paclitaxel (PTX) and curcumin (CUR), were selected for the preparation of dual-drug-loaded CUR/PTX@RSF/zein nanospheres. Both drugs achieved a high loading capacity in the RSF/zein nanospheres, i.e., 8.2% for PTX and 12.1% for CUR. Subsequently, the encapsulated PTX and CUR were released from the RSF/zein nanospheres in a sustained manner for at least 7 days. Importantly, these dual-drug-loaded RSF/zein nanospheres exhibited a considerable synergistic therapeutic effect, showing more efficient suppression of in vitro cancer cell growth than free PTX or CUR, a combination of free PTX and CUR, or single-drug-loaded nanospheres. Therefore, the CUR/PTX@RSF/zein nanospheres developed in this study hold great potential for combination chemotherapy in future clinical applications.

He-Chan Pian inhibits the metastasis of non-small cell lung cancer via the miR-205-5p-mediated regulation of the GREM1/Rap1 signaling pathway.

BACKGROUND: He-Chan Pian (HCP), a traditional Chinese medicinal formula, shows promising efficacy for the treatment of lung cancer. PURPOSE: Gremlin (GREM1) plays an important role in gastrointestinal tumor metastasis; however, little is known about its role in lung cancer. We determined the mechanism underlying the protective effect of HCP against metastasis in a mouse model of non-small cell lung cancer (NSCLC) and demonstrated the role of GREM1. METHODS: Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the herbal components and metabolites from the serum of HCP-treated mice. The tumor, liver, and kidney were examined histologically, and the antitumor effects and toxicity of HCP were evaluated. Levels of epithelial-mesenchymal transition (EMT)-associated transcription factors were measured using western blotting in tumors from five groups (i.e., model, HCP [L], HCP [M], HCP [H], and positive control [cisplatin, DDP]). Differentially expressed proteins and genes were identified using protein chip and sequencing analyzes, respectively. Short hairpin RNAs and overexpression plasmids were introduced into cells to evaluate the effects of GREM1. To evaluate proliferation, migration, and invasion, the expression levels of proteins involved in the Rap1 pathway and EMT were measured in vitro. Xenograft tumors with overexpression-GREM1 (OE-GREM1) in A549 cells were examined for cell proliferation. A dual-luciferase assay was performed to verify the direct interaction of GREM1 with miR-205-5p in lung cancer. RESULTS: Thirty-six ingredients and bioactive constituents detected in the serum of HCP-treated mice were identified as the key compounds involved in the inhibition of tumor growth. Animal experiments revealed that HCP significantly decreased tumor volumes and had no adverse effects on the liver or kidney or side effects. GREM1 upregulation was closely related to tumor metastasis and was regulated by miR-205-5p, as confirmed using a dual-luciferase reporter assay. OE-GREM1 promoted A549 cell migration and invasion, promoted EMT, and increased the expression of Rap1 pathway intermediaries, whereas shGREM1 had the opposite effects. Furthermore, the effects of OE-GREM1 on proliferation in the A549 xenograft mouse model were attenuated, although HCP has an inhibitory effect on tumors. CONCLUSION: Our results suggest that HCP contributes to the inhibition of NSCLC metastasis via the Gremlin/Rap1 signaling pathway regulated by miR-205-5p.

Herbal complex 'Buyang Huanwu Tang' improves motor endplate function of denervated-dependent skeletal muscle atrophy in rat.

Denervated-dependent skeletal muscle atrophy is a disease induced by skeletal muscle associated peripheral neuro-disconnection. Its specific molecular mechanisms remain unknown. The treating for denervated-dependent skeletal muscle atrophy is applied with an herbal complex Buyang Huanwu Tang used in traditional Chinese medicine and subjected to the established denervated-dependent skeletal muscle atrophy in rat models, and the therapeutic effects and associated mechanisms were evaluated in the pathogenesis of denervated-dependent skeletal muscle atrophy. Denervated-dependent skeletal muscle atrophy in rats was established and randomly divided into eight groups, including Normal control, Model, Positive control, Model + Buyang Huanwu Tang, Model + astragalus extracts, Model + Buyang Huanwu Tang-astragalus, Buyang Huanwu Tang + LY294002, and astragalus extract + LY294002 group. Hematoxylin-eosin staining and quantitative RT-PCR (qRT-PCR) assay were used to examine the inflammatory response of muscle tissues. Quantitative RT-PCR and Western blotting assay were utilized to analyze mRNA and protein expression. Immunohistochemistry assay was used to detect molecule expression in anterior cervical muscle tissues. Motor endplate activity was examined using the wholemount acetylcholinesterase staining method. The wet mass ratio of anterior cervical muscle was measured. The results indicated that Buyang Huanwu Tang treatment significantly alleviated inflammatory response, enhanced acetylcholinesterase activity, and motor endplate functions, and promoted wet mass of anterior cervical muscle compared to denervated-dependent skeletal muscle atrophy rat models (P < 0.05). Buyang Huanwu Tang regulated molecules of PI3K/PKB/GSK3ß/FOXO1 signaling pathway. Buyang Huanwu Tang significantly reduced muscle atrophy F-box protein, MuFR-1, Bax and caspase 9 expression, significantly enhanced Bcl-2 expression, and remarkably increased element-binding protein and vascular endothelial growth factor levels, compared to Model group (P < 0.05). Buyang Huanwu Tang suppressed caspase 9 and caspase 3 activity and associated apoptosis. Moreover, PI3K specific blocker, LY294002, significantly inhibited the effects of Buyang Huanwu Tang on the above molecule expression (P < 0.05). In conclusion, Buyang Huanwu Tang improved motor endplate functions of denervated-dependent skeletal muscle atrophy rat model through suppressing mitochondria-mediated apoptosis and activating PI3K/PKB/FOXO1 signaling pathway.

Repeated intravitreal injections of antivascular endothelial growth factor in patients with neovascular age-related macular degeneration may increase the risk of ischemic optic neuropathy.

BACKGROUND: Previous case reports have demonstrated the occurrence of ischemic optic neuropathy (ION) following intravitreal injections of antivascular endothelial growth factor (anti-VEGF). However, no previous studies have investigated the impact of injection numbers on the risk of ION. The aim of our study was to investigate whether repeated intravitreal injections of anti-VEGF would increase the risk of subsequent ION in patients with neovascular age-related macular degeneration (AMD). METHODS: A population-based, retrospective cohort study using the Taiwan National Health Insurance Research Database was conducted from 2007 to 2013. Neovascular AMD patients receiving intravitreal injections of anti-VEGF during the study period were enrolled in the study cohort. Enrollees were divided into three groups according to the categorized levels of injection number (first level: < 10 times, second level: 10-15 times, and third level: > 15 times). Kaplan-Meier curves were generated to compare the cumulative hazard of subsequent ION among the three groups. Cox regression analyses were used to estimate crude and adjusted hazard ratios (HRs) for ION development with respect to the different levels of injection numbers. The confounders included for adjustment were age, sex, and comorbidities (diabetes, hypertension, hyperlipidemia, ischemic heart disease, and glaucoma). RESULTS: In total, the study cohort included 77,210 patients. Of these, 26,520, 38,010, and 12,680 were in the first-, second-, and third-level groups, respectively. The Kaplan-Meier method revealed that the cumulative hazards of ION were significantly higher in those who had a higher injection number. After adjusting for confounders, the adjusted HRs for ION in the second- and third-level groups were 1.91 (95% confidence interval [CI], 1.32-2.76) and 2.20 (95% CI, 1.42-3.43), respectively, compared with those in the first-level group. CONCLUSIONS: Among patients with neovascular AMD, those who receive a higher number of anti-VEGF injections have a significantly higher risk of developing ION compared with individuals who receive a lower number of injections.

Buyang Huanwu Tang improves denervation-dependent muscle atrophy by increasing ANGPTL4, and increases NF-κB and MURF1 levels.

Denervated-dependent skeletal muscle atrophy (DSMA) is a disorder caused by the peripheral neuro­disconnection of skeletal muscle. The current study aimed to investigate the molecular mechanism and potential therapeutic strategies for the DSMA. A DSMA rat model was established. A lentiviral vector expressing small interfering RNA (siRNA) targeting angiopoietin­like protein 4 (ANGPTL4) was generated and injected into the rats that were also treated with Buyang Huanwu Tang (BYHWT). Reverse transcription­quantitative polymerase chain reaction was performed to examine ANGPTL4 mRNA expression in anterior cervical muscle samples. Western blot assay was used to evaluate ANGPTL4, nuclear factor­κB (NF­κB) and muscle RING­finger protein­1 (MURF1) expression. The ultrastructure of muscle tissues was viewed using transmission electron microscopy. The cell apoptosis in muscle tissues was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling. The results indicated that BYHWT treatment increased ANGPTL4 mRNA and protein levels in muscle tissues. The suppression of ANGPTL4 using siRNA significantly increased inflammatory cells compared with the control siRNA group. BYHWT protected the ultrastructure muscle tissues and inhibited cell apoptosis in the DSMA model. The protective effect of BYHWT protected may be mediated by increased expression of NF­κB p65 and MURF1. In conclusion, BYHWT may improve denervation­dependent muscle atrophy by increasing ANGPTL4 expression, involving NF­κB and MURF1 signaling.

Preventive Effects of Ginseng Total Saponins on Chronic Corticosterone-Induced Impairment in Astrocyte Structural Plasticity and Hippocampal Atrophy.

To further explore the underlying antidepressant mechanism of ginseng total saponins (GTS), this study observed the effects on hippocampal astrocyte structural plasticity and hippocampal volume in the corticosterone-induced mouse depression model. Corticosterone (20 mg/kg/day) was administered subcutaneously for 5 weeks, and GTS (12.5, 25, and 50 mg/kg/day; namely GTSL, GTSM, and GTSH) or fluoxetine (10 mg/kg/day) were given intragastrically during the last 3 weeks. On day 33 and day 34, depression-like behavior was observed via a forced swimming test and a tail suspension test, respectively. At 6 h after the last dose of corticosterone (day 35), all mice were sacrificed followed by serum corticosterone assays, stereological analysis of hippocampal glial fibrillary acidic protein-positive (GFAP+ ) astroctyes and hippocampal volume, and hippocampal glycogen tests. Results showed that all doses of GTS ameliorated depression-like behavior and the decrease in hippocampal glycogen without normalizing hypercortisolism. Moreover, GTSH and GTSM reversed the corticosterone-induced reduction in the total number of hippocampal GFAP+ astrocytes and hippocampal volume. Additionally, GTSH alleviated the diminished protrusion length and somal volume of GFAP+ astrocytes induced by corticosterone. These findings imply that the effects of GTS on corticosterone-induced depression-like behavior may be mediated partly through the protection to hippocampal astrocyte structural plasticity. Copyright © 2017 John Wiley & Sons, Ltd.

[Relationship between seedling grade of Codonopsis pilosula and yield and quality of medicinal materials].

The study is aimed to research the relationship between the seedling grade of Codonopsis pilosula and yield and quality of medicinal materials, so as to provide basis for establishing seedling standard. Thirty seedlings of C. pilosula were collected from the main production areas in Gansu province, such as Weiyuan, Minxian, Zhangxian, Dangchang and Longxi, root length and diameter and weight of all the samples were measured. According to the clustering results, seedlings were divided into 3 levels, and field experiments were conducted with three levels seedling, yield and quality were tested in laboratory. Results have showed that emergence of grades 1 was faster than that of grades 2 and 3. Yield of grades 1 was significantly higher than that of grades 2 and 3 (P<0.05). Propargyl glycoside content of grades 1 was the highest, and significantly higher than that of grades 3. Polysaccharide content of grades 3 was the highest and significantly higher than that of grades 1 and 2 (P<0.05). So considering yield, quality and investment cost of C. pilosula, planting seedlings of C. pilosula should select that root length>15.6 cm, root diameter>2.7 mm, root weight>0.56 g.

Preventive effects of ginsenoside Rg1 on post-traumatic stress disorder (PTSD)-like behavior in male C57/B6 mice.

We investigated the preventive effects of Rg1 on a model of mouse post-traumatic stress disorder (PTSD) induced by electric shock combined with situation reminder and explored the underlying mechanism. In the experiment, before the PTSD animal model was developed, Rg1 (10, 5, and 2.5mg/kg) was orally administered for one week. After the animal model was established, PTSD-like behavior was observed using elevated plus maze, black and light box, and open field tests. One hour after the behavior test, all mice were sacrificed, and then serum corticosterone (CORT) and hypothalamus corticotrophin-releasing hormone (CRH) assays were performed. Results showed that Rg1 (5mg/kg) treatments relieved PTSD-like behavior by altering elevated serum corticosterone and hypothalamus CRH levels. By contrast, fluoxetine (3mg/kg) treatment reversed the behavior changes and had no effect on increased CORT and CRH levels. These findings confirmed the preventive effect of Rg1 in PTSD model. Decreasing CORT and CRH levels may be one of the underlying mechanisms.

NRSF is an essential mediator for the neuroprotection of trichostatin A in the MPTP mouse model of Parkinson's disease.

Neuron-restrictive silencer factor (NRSF) blocks the expression of many neuronal genes in non-neuronal cells and neural stem cells. There is growing body of evidence that NRSF functions in mature neurons and plays critical roles in various neurological disorders. Our previous study demonstrated that the expression of NRSF target genes brain-derived neurotrophic factor (BDNF), and tyrosine hydroxylase (TH) is transiently decreased in 1-methyl-4-phenyl-pyridinium ion (MPP+)-treated SH-SY5Y cells. NRSF neuronal deficient mice are more vulnerable to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Here we investigated the effect of epigenetic modulation on the expression of NRSF target genes in in vitro and in vivo models of Parkinson's disease (PD). Trichostatin A (TSA) was further used to study the effects of histone deacetylase inhibition on NRSF-mediated repression. We found that the repression of NRSF target genes was relieved by TSA in vitro. A single dose TSA pretreatment also upregulated the expression of TH and BDNF and protected the nigrostriatal dopaminergic pathway against MPTP-induced degeneration in wild type mice. However, the protective functions of TSA were fully abolished in NRSF neuronal deficient mice. Our results suggest that NRSF serves as an essential mediator for the neuroprotection of TSA in the MPTP model of PD.

The antidepressant effects of ginseng total saponins in male C57BL/6N mice by enhancing hippocampal inhibitory phosphorylation of GSK-3ß.

Ginseng total saponins (GTS) are principal bioactive ingredients of Panax ginseng. In this study, we investigated the antidepressant effect of GTS on the corticosterone-induced mouse depression model and explored the underlying mechanism. Corticosterone (20 mg kg(-1) d(-1)) was subcutaneously administered for 22 d to induce the model, before doses of GTS (12.5, 25, and 50 mg kg(-1) d(-1)) or fluoxetine (10 mg kg(-1) d(-1)) were subsequently given by gavage. On day 20 and 21, depression-like behavior was observed via a forced swimming test and a tail suspension test respectively. At 6 h after the last dose of corticosterone (day 22), all mice were sacrificed followed by serum corticosterone assays and Western blot analysis. The results showed that GTS (25 and 50 mg kg(-1) d(-1))treatments relieved depression-like behavior without altering the elevated serum corticosterone levels. Furthermore, GTS treatments raised the down-regulated levels of hippocampal glycogen synthase kinase-3ß (GSK-3ß) inhibitory phosphorylation. In contrast, fluoxetine (10 mg kg(-1) d(-1)) treatment reversed the increased corticosterone level and had no effect on the decreased GSK-3ß inhibitory phosphorylation. These findings confirmed the antidepressant effect of GTS in the corticosterone-induced mouse depression model. Enhancing GSK-3ß inhibitory phosphorylation may be one of the underlying mechanisms.

Preparation and quality assessment of high-purity ginseng total saponins by ion exchange resin combined with macroporous adsorption resin separation.

AIM: To prepare high-purity ginseng total saponins from a water decoction of Chinese ginseng root. METHOD: Total saponins were efficiently purified by dynamic anion-cation exchange following the removal of hydrophilic impurities by macroporous resin D101. For quality control, ultrahigh-performance liquid chromatography with a charged aerosol detector (CAD) was applied to quantify marker components. The total saponin content was estimated by a colorimetric method using a vanillin-vitriol system and CAD response. RESULTS: D201, which consisted of a cross-linked polystyrene matrix and -N(+)(CH3)3 functional groups, was the best of the four anion exchange resins tested. However, no significant difference in cation exchange ability was observed between D001 (strong acid) and D113 (weak acid), although they have different functional groups and matrices. After purification in combination with D101, D201, and D113, the estimated contents of total saponins were 107% and 90% according to the colorimetric method and CAD response, respectively. The total amount of representative ginsenosides Re, Rd, Rg1, and compound K was approximately 22% based on ultrahigh-performance liquid chromatography-CAD quantitative analysis. CONCLUSION: These findings suggest that an ion exchange resin, combined with macroporous adsorption resin separation, is a promising and feasible purification procedure for neutral natural polar components.

Ginseng Total Saponins Reverse Corticosterone-Induced Changes in Depression-Like Behavior and Hippocampal Plasticity-Related Proteins by Interfering with GSK-3 ß -CREB Signaling Pathway.

This study aimed to explore the antidepressant mechanisms of ginseng total saponins (GTS) in the corticosterone-induced mouse depression model. In Experiment 1, GTS (50, 25, and 12.5 mg kg(-1) d(-1), intragastrically) were given for 3 weeks. In Experiment 2, the same doses of GTS were administrated after each corticosterone (20 mg kg(-1) d(-1), subcutaneously) injection for 22 days. In both experiments, mice underwent a forced swimming test and a tail suspension test on day 20 and day 21, respectively, and were sacrificed on day 22. Results of Experiment 1 revealed that GTS (50 and 25 mg kg(-1) d(-1)) exhibited antidepressant activity and not statistically altered hippocampal protein levels of brain-derived neurotrophic factor (BDNF) and neurofilament light chain (NF-L). Results of Experiment 2 showed that GTS (50 and 25 mg kg(-1) d(-1)) ameliorated depression-like behavior without normalizing hypercortisolism. The GTS treatments reversed the corticosterone-induced changes in mRNA levels of BDNF and NF-L, and protein levels of BDNF NF-L, phosphor-cAMP response element-binding protein (Ser133), and phosphor-glycogen synthase kinase-3 ß (Ser9) in the hippocampus. These findings imply that the effect of GTS on corticosterone-induced depression-like behavior may be mediated partly through interfering with hippocampal GSK-3 ß -CREB signaling pathway and reversing decrease of some plasticity-related proteins.

Preventive action of Panax ginseng roots in hypercortisolism-induced Impairment of hippocampal neurons in male C57BL/6N mice.

An increasing number of people suffering from hypercortisolism are at risk of developing hippocampus impairment and mental disorders. The aim of this study was to investigate whether the water extract of Panax ginseng roots (GWE) could prevent hypercortisolism-induced adverse consequences. Hypercortisolism was experimentally induced by repeated corticosterone injection in male mice. Treatment with corticosterone alone resulted in a significant decrease in hippocampus neurofilament light chain (NF-L) protein expression and induced depression-like behavior. Serum corticosterone was significantly increased in the corticosterone-treated mice. Treatment with GWE (800 and 400 mg/kg) during corticosterone treatment reduced or partially antagonized the effects induced by corticosterone toward the normal values of the controls; however, it failed to normalize increased corticosterone levels in corticosterone-treated mice. Overall, ginseng conclusively exhibited a protective action against hypercortisolism-induced impairment of hippocampal neurons.

Effects of sodium tanshinone II A sulphonate on hypoxic pulmonary hypertension in rats in vivo and on Kv2.1 expression in pulmonary artery smooth muscle cells in vitro.

AIM OF THE STUDY: To investigate the effect of sodium tanshinone IIA sulphonate (STS), a water-soluble derivative of tanshinone II A, on hypoxic pulmonary hypertension (HPH) in rats and its underlying mechanisms. MATERIALS AND METHODS: Rats were exposed to hypoxia for two or three weeks, pretreated with or without STS. We detected mean pulmonary arterial pressure (mPAP), the ratio of right ventricle weight to left ventricle with septum weight [RV/(LV+S)], wall thickness and voltage-activated potassium channel (Kv) 2.1 mRNA level of pulmonary arteries (PAs), respectively, and the in vitro effects of STS on proliferation and Kv2.1 expression of cultured pulmonary smooth muscle cells (PASMCs) from normal rats. Cell proliferation was determined by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazoliumbromiede (MTT) assay and direct cell counting. Kv2.1 mRNA and protein level were evaluated by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS: Chronic hypoxia increased values of mPAP and RV/(LV+S) and inhibited Kv2.1 mRNA level in PAs. Three weeks' daily STS pretreatment inhibited the hypoxia-induced increased mPAP and RV/(LV+S), pulmonary arterial thickening and up-regulated Kv2.1 mRNA level in PAs. Further study in vitro showed that STS suppressed significantly hypoxia-induced PASMCs proliferation and inhibition of Kv2.1 expression in PASMCs. CONCLUSIONS: STS might play protective effects on HPH through decreasing mPAP, V/(LV+S) and inhibiting structural remodeling in distal PAs. The mechanism of these effects may be attributed to inhibiting PASMCs proliferation and stimulating Kv2.1 expression.

Tanshinone IIA reduces lethality and acute lung injury in LPS-treated mice by inhibition of PLA2 activity.

Tanshinone IIA (TIIA) is one of the main active components from Chinese herb danshen. Previous reports showed that TIIA reduced the production of pro-inflammatory mediators stimulated with lipopolysaccharide (LPS). However, the effects of TIIA on LPS-induced acute lung injury are not fully understood. Here, we observed the effects of TIIA on mortality and lung injury in LPS-treated mice and on LPS-induced pulmonary epithelial cell injury, and further studied the underlying mechanism. As revealed by survival study, pretreatment with TIIA reduced mortality of mice and prolonged their survival time. Meanwhile, TIIA pretreatment significantly improved LPS-induced lung histopathologic changes, decreased lung wet-to-dry and lung-to-body weight ratios, inhibited lung myeloperoxidase activity and reduced protein leakage. TIIA also alleviated LPS-induced pulmonary epithelial cell injury, as proved by methyl thiazolyl tetrazolium (MTT) and lactic dehydrogenase assay. Furthermore, TIIA suppressed LPS-induced phospholipase A2 (PLA2) activity in both lung homogenate and bronchoalveolar lavage fluid. TIIA also inhibited the metabolites of PLA2, which was confirmed by results of thromboxane B2, prostaglandin E2 and leukotriene B4 detection. Besides, TIIA in vitro inhibited LPS-induced PLA2 activity in a dose-dependent manner. Western blotting showed that TIIA markedly inhibited the activation of nuclear factor kappa B (NF-kappaB) in LPS-treated mice. Taken together, these data firstly provided the novel information that the protective role of TIIA against LPS-induced lung injury may attribute partly to the inhibition of PLA2 activity and NF-kappaB activation.

[An experimental study on distribution of musk into the brain through blood brain barrier].

OBJECTIVE: To study the possible pathway of the effect of musk on brain disorder, distributing into the brain through blood brain barrier. METHODS: We used the musk ketone (muscone), a main composition of musk, to inject through the tail vein of the rats into the blood and took the brain and other organs at different times to make samples. Then gas chromatography was used to measure the distribution of muscone in the brain and other organs. RESULTS: Muscone could pass through the normal rat's blood brain barrier into the brain and soon reached the highest peak and remained in higher concentration, and more slowly metabolized as compared with other organs. CONCLUSION: Musk distributing into the brain through blood brain barrier provides the basis for its effect in treating brain disorders. Chromatography is an effective method to study the active composition of Chinese herbal medicine distributing through the blood brain barrier into the brain.