A novel mechanism for regulating lung immune homeostasis: Zukamu granules alleviated acute lung injury in mice by inhibiting NLRP3 inflammasome activation and regulating Th17/Treg cytokine balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a severely acute lung inflammation with high morbidity and mortality. Zukamu granules (ZKMG) is one of the Uygur patent drugs commonly used in clinic, which is included in the National Essential Drugs List (2018 edition). Clinical studies have shown that ZKMG has a significant effect on acute upper respiratory tract infection, and has better anti-inflammatory and antipyretic effects. However, the immunomodulatory mechanism of ZKMG on ALI is still not clear. AIM OF THE STUDY: The aim of this study is to investigate the lung protective effect and immunomodulatory mechanism of ZKMG on lipopolysaccharide (LPS) -induced ALI mice, and to provide an important basis for the treatment strategy and theoretical basis of ALI. MATERIALS AND METHODS: First, network pharmacology was used to predict the potential signaling pathways and biological processes of ZKMG related to immunology. Molecular docking technique was used to predict the possibility between the core components of ZKMG acting on NLRP3 protein. In addition, protein levels of F4/80 in lung tissues were assessed by Immunohistochemistry (IHC). The contents of IL-1ß, IL-18, IL-17A and IL-10 in the lung tissue and serum, MPO in the lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR analysis (RT-qPCR) was used to detect NLRP3 mRNA in lung tissue. Protein levels of NLRP3, Caspase-1, Cleaved caspase-1 p20, ASC, and GSDMD were detected by Western blot (WB). RESULTS: The results of network pharmacology showed that the immune pathways of ZKMG were mainly Th17 signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, etc. Molecular docking results showed that the core components of ZKMG had good binding ability to NLRP3 protein. The verification experiments showed that ZKMG can reduce the degree of lung injury, and reduce the level of inflammatory infiltration of neutrophils and macrophages by reducing the content of MPO and F4/80. In addition, ZKMG can reduce NLRP3 mRNA, inhibit the expression of NLRP3/Caspase-1/GSDMD and other related pathway proteins, and reduce inflammatory factors such as IL-1ß and IL-18. It can also reduce the content of pro-inflammatory cytokine IL-17A, increase the content of anti-inflammatory cytokine IL-10 in lung tissue. CONCLUSION: ZKMG can reduce the degree of lung tissue injury in ALI by inhibiting NLRP3/Caspase-1/GSDMD signaling pathway and restoring the IL-17A/IL-10 cytokine balance, and its protective mechanism may be related to the regulation of lung immune homeostasis. It will provide a new strategy for studying the regulation of lung immune homeostasis.

Application of network composite module analysis and verification to explore the bidirectional immunomodulatory effect of Zukamu granules on Th1 / Th2 cytokines in lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Zukamu granules (ZKMG), as the preferred drug for the treatment of colds in Uygur medical theory, has been used for 1500 years. It is also widely used in China and included in the National Essential Drugs List (2018 edition). It has unique anti-inflammatory, antitussive and analgesic effects. AIM OF THE STUDY: Aiming at the research of traditional Chinese medicine (TCM) with the characteristics of overall regulation of body diseases and the immune regulation mechanism with the concept of integrity, this paper put forward the integrated application of network composite module analysis and animal experiment verification to study the immune regulation mechanism of TCM. MATERIALS AND METHODS: The active components and targets of ZKMG were predicted, and network module analysis was performed to explore their potential immunomodulatory mechanisms. Then acute lung injury (ALI) mice and idiopathic pulmonary fibrosis (IPF) rats were used as pathological models to observe the effects of ZKMG on the pathological conditions of infected ALI and IPF rats, determine the contents of Th1, Th2 characteristic cytokines and immunoglobulins, and study the intervention of GATA3/STAT6 signal pathway. RESULTS: The results of network composite module analysis showed that ZKMG contained 173 pharmacodynamic components and 249 potential targets, and four key modules were obtained. The immunomodulatory effects of ZKMG were related to T cell receptor signaling pathway. The validation results of bioeffects that ZKMG could carry out bidirectional immune regulation on Th1/Th2 cytokines in the stage of ALI and IPF, so as to play the role of regulating immune homeostasis and organ protection. CONCLUSIONS: The network composite module analysis and verification method is an exploration to study the immune regulation mechanism of TCM by combining the network module prediction analysis with animal experiments, which provides a reference for subsequent research.

A novel therapeutic approach for IPF: Based on the "Autophagy - Apoptosis" balance regulation of Zukamu Granules in alveolar macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Zukamu Granules (ZKMG) is one of the representative Uygur patent drugs widely used in China, which is included in the National Essential Drugs List (2018 edition). As the first choice for common cold treatment in Uygur medicine theory, it has unique anti-inflammatory and antitussive efficacy. AIM OF THE STUDY: According to the recent inflammatory hypothesis, the abnormal proliferation, autophagy and apoptosis process of lung cells especially alveolar macrophages (AMs) may play an important role in the progress of idiopathic pulmonary fibrosis (IPF). Therefore, we came up with a novel treatment approach for IPF by regulating the balance of AMs "autophagy - apoptosis", and took ZKMG as the sample drug for our research. MATERIALS AND METHODS: Network pharmacology approach was conducted to predict the active components and intersected targets between ZKMG and inflammation. PPI network, GO and KEGG enrichment analysis were screened and analyzed to predict the anti-inflammatory mechanism of ZKMG. Biological experiment adopted from 128 rats, and hematoxylin-eosin staining, flow cytometry and RT-PCR were performed to examine the pathological morphology, HYP contents in lung tissue, AMs counting, AMs apoptosis, AMs phagocytosis rate, mRNA relative quantity determination of 3 key factors associated with AMs "autophagy - apoptosis" and mRNA relative quantity determination of AMs surface receptor signaling pathway. RESULTS: The predicted results showed that the mechanism of ZKMG in anti-inflammatory was related to the response and elimination of inflammatory stimuli, the intervention of apoptosis and surface receptor signaling pathways of cells. The verification experiments showed that excessive apoptosis and insufficient autophagy of AMs always existed in the progression of IPF. ZKMG could inhibit AMs proliferation, significantly reduce AMs apoptosis rate, intervene the binding of the Bcl-2 to Beclin 1, inhibit the Caspase 3 activation, stimulate the enhancement of AMs phagocytosis, and inhibit the high expression of TLR4/MyD88/NF-κB surface receptor signaling pathway, which may partly retard the fibrosis process. CONCLUSION: By inhibiting proliferation, enhancing phagocytosis, inhibiting the formation of Bcl-2 complex, and inhibiting the high expression of MYD88-dependent TLR4 signaling pathway, ZKMG can regulate the balance of AMs "autophagy - apoptosis" in the alveolitis stage to retard the fibrosis process partly. With a comprehensive strategy of "target prediction - experimental verification", we have demonstrated that inhibiting the apoptosis and promoting autophagy activity of AMs may suggest a new perspective for IPF treatment, which would provide reference for the subsequent development.

Hyperhomocysteinemia Induces Rat Memory Impairment Via Injuring Hippocampal CA3 Neurons and Downregulating cAMP Response Element-Binding Protein (CREB) Phosphorylation.

Accumulated evidence demonstrated that an elevated plasma homocysteine level, hyperhomocysteinemia, induced cognitive impairment in animals, elderly and the patients with neurodegenerative diseases. To date, the underlying cellular and molecular mechanisms by which hyperhomocysteinemia induces cognitive impairment has not been clearly defined. The purpose of this study was to investigate the possible cellular and molecular mechanisms behind hyperhomocysteinemia signaling in rat memory impairment. The results from this study demonstrated that hyperhomocysteinemia induced neuronal damage and loss in hippocampal CA3 region and downregulated the cAMP response element-binding protein (CREB) phosphorylation. The findings of this study provide evidence that hyperhomocysteinemia induces rat memory impairment via injuring hippocampal CA3 neurons and downregulating CREB phosphorylation.

HPLC-DAD analysis of Hyssopus Cuspidatus Boriss extract and mensuration of its antioxygenation property.

BACKGROUND: Hyssopus cuspidatus Boriss has been used as an important ethnomedicinal plant for long to eliminate phlegm, relieve cough and as well as having antibacterial, antioxygenation, and antitumor activities. In this study, the polyphenol contents, flavonoid contents, free radical scavenging assay and animal antioxygenation property assay of ethanol extract of H. cuspidatus were measured. METHODS: This study determined the total polyphenol and flavonoid contents in H. cuspidatus by UV-VIS. Caffeic, ferulic, and rosmarinic acids were measured using HPLC-DAD. Free radical scavenging assay of H. cuspidatus was studied by colorimetric method. Animal antioxygenation property assay of H. cuspidatus was studied with mice by biochemical assay kits. RESULTS: The total polyphenol and flavonoid contents of H. cuspidatus in 2017, 2018, 2019 were determined and the contents of H. cuspidatus in 2019 was the highest. In addition, rosmarinic acid was the phenolic acid with the highest content in H. cuspidatus. Compared with those of DPPH free radical, hydroxyl free radical, and superoxide anion free radical, the scavenging ability of H. cuspidatus of ABTS free radical was stronger, the average IC50 value was 0.0245 mg/mL. In animal antioxygenation property experiment, the model group was successfully established with decreased activities of SOD, CAT, and GSH-px and increased content of MDA. The ethanol extract of H. cuspidatus increased the activities of SOD, CAT, and GSH-px and reduced the content of MDA. Each group of samples and the ascorbic acid positive control group showed significant differences in the results of free radical scavenging and animal antioxygenation property experiments (P < 0.05). CONCLUSIONS: These results suggest that H. cuspidatus exerts an antioxygenation property, which can be attributed to the contents of total polyphenol and flavonoid. Given its strong antioxygenation property, H. cuspidatus can be used as a new natural antioxidant in food preservation and disease treatment.

Evaluation of total phenol and flavonoid content and antimicrobial and antibiofilm activities of Trollius chinensis Bunge extracts on Streptococcus mutans.

Dental caries is a chronic disease with multiple bacterial infections, Streptococcus mutans is the main cariogenic bacteria. Trollius chinensis Bunge is a common folk medicine in the Xinjiang area of China. In this study, we investigated the total flavonoid content and total phenol content in four types of T. chinensis Bunge extracts and the inhibitory effects of these extracts on S. mutans. Agar diffusion method was used to measure the inhibition zone diameters, and the minimum inhibitory concentration and minimum bactericidal concentration were determined by the twofold dilution method. Water extracts from T. chinensis Bunge and ethanol (30, 60, and 90%) extracts at different concentrations could significantly inhibit the growth of S. mutans. Among them, 30% ethanol extract exhibited the best antibacterial and antibiofilms effect. Biofilm research (crystal violet staining and CLSM) showed that 30% ethanol extract of T. chinensis Bunge plays an important role in inhibiting S. mutans growth and the number of biofilms. The results indicate that T. chinensis Bunge extract has good antibacterial and anti-biofilm activity on S. mutans. It has the potential to be developed for the treatment of caries in clinical application.

The reproductive inhibitory effects of levonorgestrel, quinestrol, and EP-1 in Brandt's vole (Lasiopodomys brandtii).

BACKGROUND: Rodent pests can inflict devastating impacts on agriculture and the environment, leading to significant economic damage associated with their high species diversity, reproductive rates and adaptability. Fertility control methods could indirectly control rodent pest populations as well as limit ecological consequences and environmental concerns caused by lethal chemical poisons. Brandt's voles, which are common rodent pests found in the grasslands of middle-eastern Inner Mongolia, eastern regions of Mongolia, and some regions of southern Russia, were assessed in the present study. METHODS: We evaluated the effects of a 2-mg/kg dose of levonorgestrel and quinestrol and a 1:1 mixture of the two (EP-1) on reproductive behavior as well as changes in the reproductive system, reproductive hormone levels, and toxicity in Brandt's voles. RESULTS: Our results revealed that all three fertility control agents can cause reproductive inhibition at a dosage of 2 mg/kg. However, quinestrol caused a greater degree of toxicity, as determined by visible liver damage and reduced expression of the detoxifying molecule CYP1A2. Of the remaining two fertility control agents, EP-1 was superior to levonorgestrel in inhibiting the secretion of follicle-stimulating hormone and causing reproductive inhibition. We believe that these findings could help promote the use of these fertility control agents and, in turn, reduce the use of chemical poisons and limit their detrimental ecological and environmental impacts.

Combination Therapy of Wuweizi (Schisandrae Chinensis Fructus) and Dexamethasone Alleviated Dexamethasone-Induced Glucocorticoid Osteoporosis in Rats with Idiopathic Pulmonary Fibrosis.

OBJECTIVE: To investigate the therapeutic effect of combined application of Wuweizi (Schisandrae Chinensis Fructus) and dexamethasone in rats with idiopathic pulmonary fibrosis (IPF) and the possible protective effect of Wuweizi against dexamethasone-induced glucocorticoid osteoporosis (GIOP). METHODS: There were five groups in this study, including the sham operation group, model group, Wuweizi group, dexamethasone group, and the combination group. A rat IPF model was made by the endotracheal injection of bleomycin. After modeling, rats were given drug interventions for 7 and 28 days. Rats were sacrificed for pathological morphology examination of the bone and lung and quantitative determination of biochemical markers of bone metabolism and angiogenesis-related cytokine to observe therapeutic efficacy on the 7th and 28th day. ELISA was used for the quantitative determination of tartrate-resistant acid phosphatase (TRACP), bone alkaline phosphatase (BALP), hypoxia-inducible factor (HIF-1α), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), and endostatin in serum. The concentrations of calcium (Ca) and phosphorus (P) were detected with the automatic biochemical analyzer. RESULTS: After drug interventions for 7 and 28 days, alveolitis and pulmonary fibrosis in treatment groups showed significant improvement compared with those in the model group (P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (α), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), and endostatin in serum. The concentrations of calcium (Ca) and phosphorus (P) were detected with the automatic biochemical analyzer. P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (P < 0.05). Bone histopathological figures showed severely damaged trabecular bone and bone marrow cavity in the dexamethasone group, but it was significantly alleviated in the combination group. The concentrations of BALP and Ca in the combination group were significantly higher than those in the dexamethasone group after treatment, while the concentrations of TRACP and P were lower than those in the dexamethasone group (α), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), and endostatin in serum. The concentrations of calcium (Ca) and phosphorus (P) were detected with the automatic biochemical analyzer. CONCLUSIONS: The combination therapy of Wuweizi and dexamethasone effectively treated IPF rats by regulating angiogenesis, meanwhile distinctly alleviating dexamethasone-induced GIOP.

In-vitro neuroprotective effect and mechanism of 2ß-hydroxy-δ-cadinol against amyloid ß-induced neuronal apoptosis.

Amyloid beta (Aß) neurotoxicity plays a causative role in the pathogenesis of Alzheimer's disease. Accumulating evidence demonstrates that Aß neurotoxicity is mediated by glutamate excitotoxicity. In our previous study, a sesquiterpenoid compound 2ß-hydroxy-δ-cadinol (HOC) which exhibited antiglutamate excitotoxicity effect was isolated from the fruits of Alpinia oxyphylla Miquel. Based on the antiglutamate excitotoxicity effect of HOC, in this study, we investigated the potential benefit of HOC in preventing Aß(1-42)-induced neuronal apoptosis in cultured rat hippocampal neurons. The neuroprotective effect of HOC against Aß(1-42)-induced neuronal apoptosis was assessed by Hoechst 33258 staining, reactive oxygen species (ROS) production, caspase-3 activation and caspase-3 activity. Results demonstrated that HOC treatment significantly prevented Aß(1-42)-induced neuronal apoptosis. The underlying molecular mechanisms of HOC in preventing Aß(1-42)-induced neuronal apoptosis may be via inhibiting Aß(1-42)-induced ROS production, attenuating Aß(1-42)-induced caspase-3 activation and inhibiting caspase-3 activity. This study suggests that HOC may be a potential agent for the prevention of Aß neurotoxicity.

[Process of gallnut suppository preparation].

The main objective was to research the process of gallnut suppository preparation with its water extract as the main drug, and evaluate its irritation to rectal mucosa. gallnut extract was obtained by decocting method, and its suppository preparation was obtained by fusion method with semi-synthetic aliphatic esters and rose flower oil as the matrix. Weight difference and in vitro melting time limit of the suppository were assayed and UV-Vis was used to determine the contents of polyphenols, tannin and saccharide. The irritation to colon mucosa was evaluated after successive administration of 14 days to New Zealand white rabbits. Finally, the prescription compositions were determined: semi-synthetic aliphatic esters and rose flower oil with the ratio of 2:1 as the proper matrix, with the drug loading of 54%. The prepared suppository was brown, conical and smooth. The weight difference was (1.43±0.03) g, with an average melting time limit of (17±2) min. The Contents of Polyphenols, tannic and polysaccharide were 332.4, 245.0, 3.3 mg•g-1 respectively in each suppository. The results also showed that the continuous administration had no irritation to rectal mucosa. It can be concluded that the suppository was an acceptable administrate form, whose preparation process was easily controlled, and with no irritation to rectum mucosa.

Neuroprotective effect and mechanism of daucosterol palmitate in ameliorating learning and memory impairment in a rat model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory decline and cognitive impairment. Amyloid beta (Aß) has been proposed as the causative role for the pathogenesis of AD. Accumulating evidence demonstrates that Aß neurotoxicity is mediated by glutamate excitotoxicity. Daucosterol palmitate (DSP), a plant steroid with anti-glutamate excitotoxicity effect, was isolated from the anti-aging traditional Chinese medicinal herb Alpinia oxyphylla Miq. in our previous study. Based on the anti-glutamate excitotoxicity effect of DSP, in this study we investigated potential benefit and mechanism of DSP in ameliorating learning and memory impairment in AD model rats. Results from this study showed that DSP administration effectively ameliorated Aß-induced learning and memory impairment in rats, markedly inhibited Aß-induced hippocampal ROS production, effectively prevented Aß-induced hippocampal neuronal damage and significantly restored hippocampal synaptophysin expression level. This study suggests that DSP may be a potential candidate for development as a therapeutic agent for AD cognitive decline.

Neuroprotective Mechanisms of 9-Hydroxy Epinootkatol Against Glutamate-Induced Neuronal Apoptosis in Primary Neuron Culture.

Glutamate-induced neuronal apoptosis has been implicated in the pathogenesis of neurological disorders. 9-Hydroxy epinootkatol (9OHEN), a sesquiterpene compound with neuroprotective activity against glutamate-induced neuronal apoptosis, was isolated from Alpinia oxyphylla Miquel in our previous study. In this study, we investigated the neuroprotective mechanisms of 9OHEN against glutamate-induced neuronal apoptosis in primary cultured neurons. The results from this study demonstrated that 9OHEN protected cortical neurons from glutamate-induced neuronal apoptosis via inhibiting glutamate-induced activation of caspase-3, inhibiting glutamate-induced reactive oxygen species (ROS) production, inhibiting glutamate-induced nitric oxide (NO) production, and downregulating glutamate-induced neuronal nitric oxide synthase (nNOS) expression. This study suggest that 9OHEN might have therapeutic potential in treating glutamate-mediated neurological diseases.

Neuroprotective effect of biatractylenolide against memory impairment in D-galactose-induced aging mice.

Biatractylenolide, a sesquiterpene lactone, which exerted the neuroprotective effect against glutamate-induced excitotoxicity, was isolated from Atractylodis macrocephala in our previous study. In this study, we evaluated the neuroprotective effect of biatractylenolide against D-galactose-induced memory impairment and explored the potential mechanism of its action. The results showed that administration of biatractylenolide could significantly improve behavioral performance of D-galactose-treated mice in passive avoidance test and spatial learning-memory test. Administration of biatractylenolide could significantly decrease the formation of reactive oxygen species (ROS), decrease the activity of acetylcholinesterase (AChE), and increase the expression of synapsin I and protein kinase C (PKC) in D-galactose-treated mice. Our findings provide first evidence for the neuroprotective effect of biatractylenolide against D-galactose-induced memory impairment. The potential mechanisms underlying the neuroprotective effect of biatractylenolide in D-galactose-treated mice might be (i) attenuating oxidative damage via decreasing ROS formation, (ii) restoring cholinergic neurotransmission via decreasing AChE activity, and (iii) increasing the expression of memory-related proteins (synapsin I and PKC). Biatractylenolide may have therapeutic potential in aging-related memory impairment.

Neuroprotection of atractylenolide III from Atractylodis macrocephalae against glutamate-induced neuronal apoptosis via inhibiting caspase signaling pathway.

Glutamate-induced excitotoxicity appears to play a crucial role in neurological disorders. Neuroprotection against glutamate-induced excitotoxicity has been proposed as a therapeutic strategy for preventing and/or treating these excitotoxicity-mediated diseases. In the present study, atractylenolide III, which exhibited significantly neuroprotective effect against glutamate-induced neuronal apoptosis, was isolated from Atractylodes macrocephala by means of bioactivity-guided fractionation. The inhibitory effect of atractylenolide III on glutamate-induced neuronal apoptosis was in a concentration-dependent manner. The anti-apoptotic property of atractylenolide III might be mediated, in part, via inhibiting caspase signaling pathway. Atractylenolide III may have therapeutic potential in excitotoxicity-mediated neurological diseases.

[Diverse expression of ER-α36, a novel variant of ER-α, in hippocampus and cortex of neonatal and adult rats].

ER-α36, a novel variant of ER-α, is expressed in breast, uterus, digestive tract, respiratory tract etc. The aim of the present study was to investigate the distribution and expression of ER-α36 in the central nervous system (CNS). Here, we comparatively analyzed the expression pattern of ER-α36 in the hippocampus and cortex of neonatal (1-day-old) and adult (12-week-old) Sprague-Dawley (SD) rats by using immunohistochemistry/immunocytochemistry analysis and Western blot. The results showed that ER-α36 was expressed both in hippocampus and cortex of adult rats, but mainly distributed in pyramidal neurons. ER-α36 was mainly located on the cytomembrane of hippocampal and cortical neurons from neonatal rats. Compared with the cortical neurons, the hippocampal neurons showed lower ER-α36 protein expression in the neonatal rats, but exhibited higher level of ER-α36 in the adult rats. Furthermore, the adult rats showed higher levels of ER-α36 expression in both hippocampus and cortex compared with the neonatal rats. These results suggest that ER-α36 might be involved in the regulation of membrane-initiated estrogen signaling throughout the postnatal development of diverse brain regions, and thus will be a potential target for the treatment of degenerative diseases in nervous system.

[Knock-down of ERα36 impacts the expression of differentiation protein in PC12 cells].

ERα36 is a novel subtype of estrogen receptor alpha (ERα) known to play an important role in breast cancer development and widely expressed in normal tissues and cells including nerve cells. However, the expression and function of ERα36 in nerve cells have not been well elucidated. To examine whether ERα36 is involved in differentiation of nerve cells, the differentiated and undifferentiated PC12 (PC12D and PC12unD) cells were used. Transfection of ERα36-shRNA plasmid into PC12 cells was performed to establish the ERα36 gene knock-down cells model. Immunocytofluorescence and Western blot were used to analyze the expression of Nestin, ß-tubulinIII and Neu-N in the PC12 cells. The results showed that ERα36 was expressed in both cell types. Compared with PC12D cells, PC12unD cells showed higher expression of Nestin and lower expression of ß-tubulinIII. ERα36-shRNA-mediated knock-down of ERα36 expression enhanced the expression of ß-tubulinIII and Neu-N, but attenuated Nestin expressions in PC12unD cells; ERα36 knock-down in PC12D cells mediated Nestin, ß-tubulinIII and Neu-N in a contrary manner. These results indicate that ERα36 knock-down appear to be associated with inhibiting differentiation in differentiated cells and promoting differentiation in undifferentiated cells, suggesting that ERα36 is a dual regulator in nerve differentiation.

Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum.

In this study, we employed TiO2 enrichment and high accuracy liquid chromatography-mass spectrometry-mass spectrometry to identify the phosphoproteome of Clostridium acetobutyicum ATCC824 in acidogenesis and solventogenesis. As many as 82 phosphopeptides in 61 proteins, with 107 phosphorylated sites on serine, threonine, or tyrosine, were identified with high confidence. We detected 52 phosphopeptides from 44 proteins in acidogenesis and 70 phosphopeptides from 51 proteins in solventogenesis, respectively. Bioinformatic analysis revealed most of the phosphoproteins located in cytoplasm and participated in carbon metabolism. Based on comparison between the two stages, we found 27 stage-specific phosphorylated proteins (10 in acidogenesis and 17 in solventogenesis), some of which were solvent production-related enzymes and metabolic regulators, showed significantly different phosphorylated status. Further analysis indicated that protein phosphorylation could be involved in the shift of stages or in solvent production pathway directly. Comparison against several other organisms revealed the evolutionary diversity among them on phosphorylation level in spite of their high homology on protein sequence level.

Neuroprotective effect of Rhizoma Atractylodis macrocephalae against excitotoxicity-induced apoptosis in cultured cerebral cortical neurons.

Excitotoxicity has been implicated in neurological disorders. This study investigated the neuroprotective effect of the extract from Rhizoma Atractylodis macrocephalae on excitotoxicity-induced neuronal apoptosis in primary cultured cerebral cortical neurons. Excitotoxicity was induced by exposure of cortical neurons to glutamate. Neuronal apoptosis and the protective effect of Rhizoma Atractylodis macrocephalae extract were examined by multi-indices including cell viability assay, morphological features, DNA fragmentation and flow cytometric analysis. After exposure of cultured neurons to glutamate for 24 h, the neurons exhibited marked apoptotic-like death. Co-treatment of the neurons with glutamate and Rhizoma Atractylodis macrocephalae extract significantly elevated the cell viability, and reduced the number of apoptotic cells. These results demonstrate that Rhizoma Atractylodis macrocephalae is an effective neuroprotective agent against glutamate-induced excitotoxicity and may have therapeutic potential in excitotoxicity-mediated diseases.