[Experimental study of cardioprotective effects of Cinnamomi Ramulus and Cinnamomi Cortex formula granules on myocardial ischemia/reperfusion injury in rats based on efficacy of "warming and coordinating heart Yang"].

This study aimed to parallelly investigate the cardioprotective activity of Cinnamomi Ramulus formula granules(CRFG) and Cinnamomi Cortex formula granules(CCFG) against acute myocardial ischemia/reperfusion injury(MI/RI) and the underlying mechanism based on the efficacy of "warming and coordinating the heart Yang". Ninety male SD rats were randomly divided into a sham group, a model group, CRFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, and CCFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, with 15 rats in each group. The sham group and the model group were given equal volumes of normal saline by gavage. Before modeling, the drug was given by gavage once a day for 7 consecutive days. One hour after the last administration, the MI/RI rat model was established by ligating the left anterior descending artery(LAD) for 30 min ischemia followed by 2 h reperfusion except the sham group. The sham group underwent the same procedures without LAD ligation. Heart function, cardiac infarct size, cardiac patho-logy, cardiomyocyte apoptosis, cardiac injury enzymes, and inflammatory cytokines were determined to assess the protective effects of CRFG and CCFG against MI/RI. The gene expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3(NLRP3) inflammasome, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate specific proteinase-1(caspase-1), Gasdermin-D(GSDMD), interleukin-1ß(IL-1ß), and interleukin-18(IL-18) were determined by real-time quantitative polymerase chain reaction(RT-PCR). The protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD were determined by Western blot. The results showed that both CRFG and CCFG pretreatments significantly improved cardiac function, decreased the cardiac infarct size, inhibited cardiomyocyte apoptosis, and reduced the content of lactic dehydrogenase(LDH), creatine kinase MB isoenzyme(CK-MB), aspartate transaminase(AST), and cardiac troponin Ⅰ(cTnⅠ). In addition, CRFG and CCFG pretreatments significantly decreased the levels of IL-1ß, IL-6, and tumor necrosis factor-α(TNF-α) in serum. RT-PCR results showed that CRFG and CCFG pretreatment down-regulated the mRNA expression levels of NLRP3, caspase-1, ASC, and downstream pyroptosis-related effector substances including GSDMD, IL-18, and IL-1ß in cardiac tissues. Western blot revealed that CRFG and CCFG pretreatments significantly decreased the protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD in cardiac tissues. In conclusion, CRFG and CCFG pretreatments have obvious cardioprotective effects on MI/RI in rats, and the under-lying mechanism may be related to the inhibition of NLRP3/caspase-1/GSDMD signaling pathway to reduce the cardiac inflammatory response.

Isolation, structure and bioactivity of polysaccharides from Atractylodes macrocephala: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Polysaccharides from Atractylodes macrocephala are important components isolated and extracted from the traditional Chinese medicine named Atractylodes macrocephala Koidz. Traditionally, A. macrocephala has been used to strengthen the spleen, benefit qi, dry dampness and promote water circulation, and prevent miscarriage. As the main components, polysaccharides from A. macrocephala have a variety of related pharmacological activities, such as the ability to regulate the gastrointestinal tract, protect the liver and so on. AIM OF THE REVIEW: This review aims to compile the extraction and purification methods, structural characteristics and pharmacological activities of polysaccharides from A. macrocephala and the mechanisms of actions to explore the future application potential of polysaccharides from A. macrocephala. MATERIALS AND METHODS: Valid and comprehensive relevant information was collected from China National Knowledge Infrastructure, Web of Science, Pubmed and so on. RESULTS: More than 20 polysaccharides have been extracted from A. macrocephala, different extraction and purification methods have been described, and the composition structures and pharmacological activities of polysaccharides from A. macrocephala have been reviewed. Polysaccharides, as important components of A. macrocephala, were mainly extracted by four methods such as water decoction, ultrasonic-assisted extraction, complex enzyme method and microwave-assisted extraction, and then were obtained through decolorization, deproteinization and separation and purification by various chromatographic columns. The chemical compositions and structures of polysaccharides from A. macrocephala show diversification, and three structural formulae have been confirmed at this stage. Polysaccharides from A. macrocephala have a variety of pharmacological activities, such as immunomodulation, antitumor, antioxidant, hepatoprotection, gastrointestinal mucosa protection, neuroprotection, hypoglycemia, growth promotion and so on. CONCLUSIONS: There is a diversity in the compositional structures of polysaccharides from A. macrocephal, which have multiple biological activities and promising applications. Therefore, further understanding of the relationship between structures and functions of polysaccharides from A. macrocephaly, and potential synergistic effects with other substances is especially important for its development and utilization.

Traditional uses, phytochemical constituents and pharmacological properties of Osmanthus fragrans: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Osmanthus fragrans Lour., is a medicinal plant distributed widely in some Asian countries including Japan and Korea and southwestern China. It has been used traditionally for the treatment of weakened vision, halitosis, panting, asthma, cough, toothache, stomachache, diarrhea, rheumatism, physique pain and hepatitis. AIM OF THE REVIEW: Recent advances in traditional uses, botanical characteristics, distribution, taxonomy, phytochemical constituents, biological effects as well as the toxicities of O. fragrans are comprehensively presented and critically evaluated, and the underlying mechanism associated with the bioactivities of extracts, essential oil and components from this plant is also well summarized. In order to provide comprehensive scientific basis for the medical application and help interested researchers discover food and medicinal natural products from O. fragrans. MATERIALS AND METHODS: All information was systematically gathered from globally accepted scientific databases by Internet databases, including Elsevier, ScienceDirect, PubMed, Web of Science, Wiley, Springer, SciFinder, ACS Publications, CNKI, WanFang, Google Scholar, Baidu Scholar, The Plant List Database, and other literature sources (Ph.D. and MSc dissertations). All published contributions on O. fragrans different languages were included and cited. The chemical structures of all isolated compounds were drawn by using ChemBioDraw Ultra 14.0 software. RESULTS: To date, more than 183 compounds were isolated and structurally identified from different plant parts of O. fragrans. Among them, ionone, ionol, flavonoids, polyphenols and iridoids, as the major bioactive substances, have been extensively studied and displayed the best bioactivity. Pharmacological studies demonstrated that O. fragrans and its active components had a wide range of biological activities, such as antioxidant, antitumor, anti-inflammatory, anti-hyperglycemic, anti-thrombotic, anti-melanogenesis, neuroprotective, and hepatoprotective activities, etc. CONCLUSION: O. fragrans, as a food and medicinal resource, has a good health care function and important edible and medicinal value, and thus has good prospects for utilization. However, many studies on biological activities were mainly based on extracts and the bioactive ingredients of this plant, and the mechanism responsible for these extracts and ingredients have not been well identified and there is a gap in research regarding clinical effect and safety. Therefore, the detail in vitro and in vivo studies on the mechanisms of action of the pure bioactive compounds and more clinical studies are encouraged to be conducted to ensure safety and effectiveness of the plant for human use.

The antitumour activity of C21 steroidal glycosides and their derivatives of Baishouwu: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Baishouwu has been used in China for thousands of years since it was first discovered in the late Tang Dynasty and flourished in the Song and Ming Dynasties. The Chinese herbal medicines named Baishouwu include Cynanchum auriculatum Royle ex Wight., Cynanchum bungei Decne. and Cynanchum wilfordii Hemsl. It is described in the Sign of Materia Medica as "sweet, bitter, reinforce liver and kidney, and non-toxic". It is widely used for nourishing the blood to expel wind, reinforcing liver and kidney, strengthening bones and muscles. AIM OF THE REVIEW: In this review, the current research status of the C21 steroidal glycosides and their derivatives of Baishouwu for malignant tumours and their anti-tumour mechanisms are discussed. This may lay the ground for potential application of Baishouwu and its active ingredients in the treatment of tumours. MATERIALS AND METHODS: Scientific databases, including PubMed, Elsevier, Science Direct, Google Scholar, CNKI, WANFANG DATA and VIP were searched to gather data about Baishouwu and its C21 steroidal glycosides and their derivatives. RESULTS: Prior literature indicates that Baishouwu has important biological activities such as anti-tumour, anti-epileptic, reducing cholesterol, protection of liver and kidney and immunomodulatory, which are of increasing interest, especially its anti-tumour activity. Recent studies demonstrate that the C21 steroidal glycosides of Baishouwu, which have prominent antitumour efficacy, are one of its main active ingredients. Presently, a variety of C21 steroidal glycosides have been isolated from Baishouwu medicinal part, the tuberous root. This review summarizes the various antitumour activities of the C21 steroidal glycosides and their derivatives of Baishouwu. CONCLUSIONS: In this review, the antitumour effects and mechanisms of total C21 steroidal glycosides and monomers and derivatives of Baishouwu in vitro and in vivo were summarized. Baishouwu can inhibit tumourigenesis by blocking tumour cell cycle progression, regulating numerous signaling pathways, promoting apoptosis, inhibiting tumour cells proliferation and metastasis, improving immunity and so on. This review provides a theoretical basis for inheriting and developing the medical heritage of the motherland, exploring the resources of traditional Chinese medicine for ethnic minorities and clinical rational drug use.

Cinnamic acid preserves against myocardial ischemia/reperfusion injury via suppression of NLRP3/Caspase-1/GSDMD signaling pathway.

BACKGROUND: Cinnamic acid (CA) is an active organic acid compound extracted from Cinnamomi ramulus that has various biological activities. There is growing studies have shown that the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome significantly contributes to sterile inflammatory response and pyroptosis in myocardial ischemia/reperfusion injury (MI/RI). However, whether CA has any influence on NLRP3 inflammasome and pyroptosis during MI/RI are not fully elucidated. PURPOSE: In the present study, we investigated whether NLRP3 inflammasome activation and pyroptosis were involved in the cardioprotective effect of CA against MI/RI. METHODS: Male Sprague-Dawley rats were intragastrically administered either with CA (75 and 150 mg/kg, daily) or vehicle for 7 successive days prior to ligation of coronary artery, and then rats were subjected to ligation of the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min to evoke MI/RI. RESULTS: Our results demonstrated that CA could significantly improve cardiac diastolic function, decrease cardiac infarct size and myocardial injury enzymes, inhibit cardiomyocyte apoptosis, attenuate cardiac structure abnormality, and mitigate oxidative stress and inflammatory response. We also found that MI/RI activate NLRP3 inflammasome as evidenced by the upregulation levels of NLRP3, pro-caspase-1, caspase-1, and ASC proteins and mRNA. More importantly, MI/RI trigger pyroptosis as indicated by increased DNA fragmentation, membrane pore formation, and mitochondrial swelling as well as increased levels of pyroptosis-related proteins and mRNA, including GSDMD, N-GSDMD, IL-18, and IL-1ß. As expected, all these deleterious alterations were prominently reversed by CA pretreatment. CONCLUSIONS: These findings indicate that CA effectively protected cardiomyocytes against MI/RI by inhibiting NLRP3/Caspase-1/GSDMD signaling pathway, and it is worthy of more investigations for its therapeutic potential for extenuating ischemic heart disease.

JFNE-A isolated from Jing-Fang n-butanol extract attenuates lipopolysaccharide-induced acute lung injury by inhibiting oxidative stress and the NF-κB signaling pathway via promotion of autophagy.

BACKGROUND: Jing-Fang powder consists of Jingjie (Nepeta tenuifolia Benth, (Lamiaceae)). and Fangfeng (Saposhnikovia divaricata (Turcz.) Schischk, (Apiaceae)) Previous studies have revealed that the Jing-Fang powder n-butanol extract (JFNE) has anti-acute lung injury (ALI) and anti-inflammatory properties; however, the active ingredient and mechanism remain unknown. PURPOSE: In the present study, we investigated the anti-inflammatory effect of a bioactive fraction obtained from JFNE(JFNE-A) on lipopolysaccharide (LPS)-induced ALI in mice and explored the underlying mechanism. STUDY DESIGN: The anti-acute lung injury effect and mechanism of JFNE-A was investigated by prophylactic administration of JFNE-A in mice with LPS-induced acute lung injury. METHODS: The expression levels of myeloperoxidase(MPO) in lung tissues of mice and interleukin(IL)-6, tumor necrosis factor(TNF)-α, IL-1ß, IL-5, interferon (IFN)-γ, monocyte chemotactic protein (MCP)-1, macrophage colony stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, and MIP-1ß in bronchi alveolar lavage fluid (BALF) were detected by reagent kit and the histological changes were examined by hematoxylin and eosin (H & E) for general histopathological conditions under a light microscope. In addition, the ultrastructure of the cells in lung tissues were observed and photographed under a transmission electron microscope. The expression levels of protein were detected via Western blotting and the mRNA expression of relative genes were determined of via reverse transcriptase polymerase chain reaction (RT-PCR). What's more, we also further clarified the potential targets of JFNE-A through network pharmacology analysis, which could be utilized in ALI treatment. RESULTS: Our results showed that pretreatment with JFNE-A for 7 days significantly reduced the lung pathological injury score, alleviated pulmonary edema, and decreased the lung tissue MPO level. Mechanistically, JFNE-A dramatically downregulated the protein levels of IL-6, TNF-α, IL-1ß, M-CSF, and IFN-γ in BALF and mRNA expression levels of IL-6, TNF-α, IL-1ß, and IFN-γ in lung tissues. JFNE-A also significantly lowered the protein levels of iNOS and phosphorylated NF-κB (p65) and mRNA expression levels of iNOS, Rela, CHUK, and NF-κB1, and also elevated the protein expression levels of Nrf2, HO-1, and SOD1 and the mRNA expression levels of Nrf2, Hmox1, and Keap-1 in the lungs. Moreover, JFNE-A significantly decreased the protein expression of p62 and increased the ratio of LC3II/LC3I. It also upregulated the mRNA expression levels of Atg5 and Beclin-1, whereas it reduced the mRNA expression level of SQSTM1 and increased autophagosome structures. CONCLUSION: Overall, treatment with JFNE-A ameliorated LPS-induced ALI in mice by suppressing the NF-κB signaling pathways and promoting Nrf2 signaling pathways by accelerating autophagy.

Xiaoyaosan ethyl acetate fraction alleviates depression-like behaviors in CUMS mice by promoting hippocampal neurogenesis via modulating the IGF-1Rß/PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoyaosan (XYS), a representative and classic traditional Chinese medicine (TCM) prescription with function of dispersing stagnated liver and strengthening spleen, has been used for thousands of years to treat depression. XYS' anti-depression effect has been demonstrated both clinically and experimentally; however, the material basis for this effect has yet to be elucidated. AIM OF THE STUDY: This study aimed to evaluate the impact and underlying action mechanism of XYS' antidepressant active component (Xiaoyaosan ethyl acetate fraction, XYSEF) against chronic unpredictable mild stress (CUMS)-induced depression-like behavior in mice. MATERIALS AND METHODS: First, we established a behavioral despair depression mouse model to preliminarily determine the effective antidepressant dose of XYSEF. Then, we created a CUMS mouse model and used various classic behavioral tests, including SPT, ST, NFST, and TST, to assess XYSEF's antidepressant properties. IGF-1 levels in mouse serum and hippocampus were quantified using ELISA. The average optical density of Nissl bodies in the mouse hippocampal CA3 region was determined utilizing toluidine blue staining. Brdu and DCX expression in the hippocampal dentate gyrus (DG) was assayed using the immunofluorescence method. IGF-1Rß, PI3K, p-PI3K, Akt, p-Akt, Caspase-3, and cleaved Caspase-3 protein levels in the hippocampus were determined with Western blot. RESULTS: The behavioral despair mouse model findings showed that 9.1 and 40 g/kg of XYSEF both significantly shortened the immobility time of mice, suggesting that the effective dose range was 9.1-40 g/kg. Compared to the CUMS mouse model, XYSEF at 20 and 40 g/kg markedly increased the sucrose preference percentage in the SPT and grooming time in the ST, shortened the immobility time in the TST and the feeding latency in the NSFT, and reversed the downregulated IGF-1 content in mouse serum and hippocampus. In addition, XYSEF amplified the average optical density of Nissl bodies in the hippocampal CA3 region, promoted Brdu and DCX expression in DG, and diminished IGF-1Rß, p-PI3K/PI3K, p-Akt/Akt, and cleaved Caspase-3/Caspase-3 protein levels in the hippocampi of CUMS mice. CONCLUSION: XYSEF acted as an antidepressant in mice exhibiting CUMS-induced depression-like behaviors, possibly by promoting hippocampal neurogenesis, reducing neuronal apoptosis, and inhibiting the over-activation of the IGF-1Rß/PI3K/Akt pathway.

[Mechanism of Jingfang Granules in relieving alcohol and protecting liver based on bioinformatics technology].

The present study explored the potential mechanism of Jingfang Granules in relieving alcohol and protecting liver by network pharmacology and molecular docking and verified the effects and related pathways by animal experiments. The active components of Jingfang Granules were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Targets of drugs and diseases were obtained from PubChem, Swiss Target Prediction and CTD. The common targets were uploaded to STRING to plot the protein-protein interaction(PPI) network. The core targets were screened out and the target organs were identified by Bio GPS and Metascape, followed by Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of common targets. The acute drunk mouse model was established and the effects of Jingfang Granules on serum ethanol level and the expression of proteins related to the phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt) signaling pathway in the liver tissue of mice were observed. A total of 187 active components of Jingfang Granules were obtained, including 47 common targets with alcoholic liver injury. GO enrichment analysis and KEGG pathway analysis showed that Jingfang Granules might play the role of relieving alcohol and protecting liver through the PI3 K-Akt signaling pathway. The drug-component-target and component-target-pathway networks revealed that the important active components of Jingfang Granules in relieving alcohol and protecting liver included quercetin, 5-O-methylvisamminol, glyasperin M, glyasperin B and hederagenin. Molecular docking showed that the active components had a good affinity with AKT1, EGFR, ESR1 and PTGS2. Experimental results showed that Jingfang Granules(15 and 10. 5 g·kg-1) could significantly reduce the content of serum ethanol in mice and up-regulate the protein expression ratios of p-PI3 K/PI3 K and p-Akt/Akt in the liver tissue. Jingfang Granules could relieve alcohol and protect liver through multi-component and multitarget, and the mechanism may be related to the activation of the PI3 K-Akt signaling pathway.

Network pharmacology and in vitro study of Fengreqing oral liquid in the intervention of wind-heat pattern.

OBJECTIVE: To investigate the underlying mechanism of the effect of Fengreqing oral liquid (, FOL) on wind-heat pattern (WHP). METHODS: In this study, we predicted the potential targets of FOL via the approach of network pharmacology and verified it by in vitro inflammation model. In the network pharmacology part, two strategies, namely the direct target search and the indirect one, were used to collect the target sets of FOL in WHP treatment. The enrichment analysis was carried out by David database and ClueGo plug-in in Cytoscape. Furthermore, the potential targets were mapped in the candidate pathways. In the verification experiment section, in vitro model of lipopolysaccharide (LPS) induced RAW 264.7 was used to confirm the predictive results in the network pharmacology part. RESULTS: Through the two screening strategies, a total of 141 non-repetitive intervention targets of FOL on WHP were obtained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the intervention effect was mainly focused on the anti-inflammatory effect, and the Toll-like receptor signaling pathway was one of the most critical regulatory pathways. Further mapping analysis showed that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling transfer might be the key part of regulating the concentration of inflammation mediators of FOL in the Toll-like receptor signaling pathway. In vitro experiment showed that FOL significantly reduced the levels of NO, IL-1, IL-6, and TNF-α produced by RAW264.7 induced by LPS. Further immunofluorescence found that this effect is related to the regulation of PI3K-AKT pathway activity by FOL. CONCLUSION: FOL can intervene in WHP by regulating the content of inflammatory mediators via the PI3K-AKT pathway.

Cardioprotective activity of ethyl acetate extract of Cinnamomi Ramulus against myocardial ischemia/reperfusion injury in rats via inhibiting NLRP3 inflammasome activation and pyroptosis.

BACKGROUND: NLRP3 inflammasome activation and pyroptosis play an important role in myocardial ischemia/reperfusion injury (MI/RI). Cinnamomi ramulus (CR), is an important folk medicinal plant in China, which derived from the dried twig of Cinnamomum cassia (L.) Presl, has function of "warming and tonifying heart yang", and traditionally utilized to treat the cold, blood-cold amenorrhea, phlegm, edema, arthralgia, and palpitations as well as improve blood circulation. The aqueous extract of C. ramulus was reported to show significant therapeutic potential for treating MI/RI. Whereas, there are no previous investigations in China or abroad has reported the cardioprotective effects and underlying mechanism of the ethyl acetate extract of C. ramulus (CREAE) and its bioactive substance cinnamic acid (CA) in triggering NLRP3 inflammasome activation and subsequent pyroptosis. PURPOSE: The present study aimed to assess the cardioprotective function of CREAE and CA against the MI/RI in rats and involved the underlying mechanisms. METHODS: The MI/RI model was established in male SD rats by occlusion of the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min, respectively. The rats were intragastrically administered with CREAE (74 and 37 mg/kg) and CA (45 mg/kg) for 7 successive days before vascular ligation. The cardioprotective effects of CREAE and CA against myocardial injury of rats were detected by HE staining, TTC staining, echocardiograms, and myocardial enzymes detections. Serum levels of inflammatory factors, such as IL-6, IL-1ß, and TNF-α, were analyzed by ELISA kits to evaluate the effects of CREAE and CA. The protein and gene expression levels of NLRP3 and the pyroptosis-related factors in heart tissue were conducted by western blot and RT-qPCR. RESULTS: Our results showed that CREAE and CA decrease myocardial infarct size and improve cardiac function, mitigate myocardial damage, and repress inflammatory response in rats after I/R. Mechanistically, our results revealed that CREAE and CA can dramatically suppress the activation of NLRP3 inflammasome and subsequent cardiomyocyte pyroptosis in myocardial tissues that as evidenced by downregulating the protein and gene expressions of NLRP3, ASC, IL-1ß, caspase-1, gasdermin D, and N-terminal GSDMD. CONCLUSIONS: Our data indicated that CREAE and CA may attenuate MI/RI through suppression of NLRP3 inflammasome and subsequent pyroptosis-related signaling pathways.

[Anti-inflammatory effect and mechanism of ethanol extract from Saposhnikoviae Radix in LPS-induced inflammation mouse model].

This paper aimed to explore the anti-inflammatory effect of ethanol extract from Saposhnikoviae Radix in a lipopolysaccharide(LPS)-induced inflammation mouse model and its regulation of TLR4/NF-κB signaling pathway. The ethanol extract from Saposhnikoviae Radix was separated and purified on the macroporous adsorption resin and its main chemical components were identified by UPLC-QE/MS. The identification results showed that the top ten components of ethanol extract from Saposhnikoviae Radix were mainly chromones and coumarins. A mouse model of inflammation induced by intraperitoneal injection of LPS was used to investigate the anti-inflammatory effects of ethanol extract from Saposhnikoviae Radix after intragastric administration for seven successive days. Mice in all groups except for the control group were treated with intraperitoneal injection of LPS(0.015 g·kg~(-1)) one hour after the last administration, and twelve hours later, the blood was sampled and separated and the broncoalveolar lavage fluid(BALF) was collected. The levels of nitric oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1ß(IL-1ß) in mouse serum and BALF were detected by ELISA. The harvested lung tissue was stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by the detection of protein expression levels of related molecules in TLR4/NF-κB signaling pathway by Western blotting. The results showed that the ethanol extract from Saposhnikoviae Radix significantly ameliorated the pathological conditions in lung tissue of model mice, reversed the increase in NO, TNF-α, IL-6, and IL-1ß levels of mouse serum and BALF, down-regulated the protein expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor(MyD88), and phosphorylated nuclear transcription factor κB-p65/nuclear transcription factor κB-p65(P-NF-κB p65/NF-κB p65), and up-regulated the NF-κB inhibitory protein α(IκBα). The ethanol extract from Saposhnikoviae Radix exhibited a good anti-inflammatory effect in the LPS-induced acute inflammation muse model, which might be related to the inhibition of the activation of TLR4/NF-κB inflammatory signaling pathway. Chromones and coumarins have been proved to be the active components for its anti-inflammatory effects.

Anti-type I allergic effects of Jing-Fang powder extracts via PI3K/Akt pathway in vitro and in vivo.

Jing-Fang powder (Schizonepeta tenuifolia Briq. and Saposhnikovia divaricata (Turcz.) Schischk.) was used to treat chronic bronchitis, asthma and chronic urticaria. Based on the preliminary results of screening research on the antiallergic effective parts of Jing-Fang powder, its ethyl acetate extract fractions (JFEE) and isolate D (JFEE-D) showed the best anti-allergic effect. RBL-2H3 cell activation degranulation model and mice passive cutaneous anaphylaxis (PCA) reaction model were used to investigate the effects and mechanisms of JFEE and JFEE-D on IgE-mediated type I allergic reactions. LC-MS was utilized to determine the composition of JFEE and JFEE-D. We found that JFEE and JFEE-D significantly reduced ß-HEX, histamine, IL-4, IL-6 levels in cell supernatants, and improved the degree and morphology of cell degranulation. JFEE and JFEE-D significantly inhibited the increase of ear vascular permeability and abnormal increase of serum IgE, TNF-α, IL-6 levels. JFEE and JFEE-D inhibited mRNA expression of PI3K and Akt and down-regulated protein expression of PI3K, Akt, p-Akt, and PLCγ1 in sensitized RBL-2H3 cells. The combined use of JFEE and JFEE-D with pathway inhibitor Wortmannin revealed synergistic down-regulation of PI3K, Akt, and p-Akt protein expression. The combined use of pathway agonist IGF-1, JFEE and JFEE-D down-regulated increase of p-Akt/Akt protein expression. Moreover, JFEE and JFEE-D significantly inhibited protein expression of PI3K, p-Akt and PLCγ1 in PCA model mice. These results show that JFEE and JFEE-D inhibit type I allergic reactions by inhibiting PI3K/Akt signaling pathway.

Xiaoyao Pills Attenuate Inflammation and Nerve Injury Induced by Lipopolysaccharide in Hippocampal Neurons In Vitro.

Lipopolysaccharides (LPS) are proinflammation mediators that can induce the inflammatory model of the hippocampal neuron, and neuroinflammation participates in the pathophysiology of depression. Xiaoyao Pill is a classical Chinese medicine formula that has been used for the treatment of mental disorders such as depression in China since the Song dynasty. We established a hippocampal neuronal cell inflammation model by LPS and investigate the intervention effect and mechanism of Xiaoyao Pills. The expression levels of IL-6, TNF-α, IDO, 5-HT, brain-derived neurotrophic factor, and ß-nerve growth factor were detected by enzyme-linked immunosorbent assay. mRNA levels of IL-6, TNF-α, 5-HT1A, IDO-1, brain-derived neurotrophic factor, nerve growth factor, tropomyosin receptor kinase B, tropomyosin receptor kinase A, and cAMP response element-binding protein were detected by reverse transcription-polymerase chain reaction. To further validate, protein expression was determined by western blot and immunofluorescence. Lipopolysaccharide-induced neuroinflammatory state resulted in the release of IL-6, TNF-α, and IDO and a decrease of BDNF, NGF, TrkB, TrkA, CREB, p-CREB, p-CREB/CREB, and SYP and inhibited hippocampal neurogenesis in the hippocampal neuron. Xiaoyao Pills significantly decreased the levels of IL-6, TNF-α, and IDO in cell supernatant and increased the expression of BDNF, NGF, TrkB, TrkA, CREB, p-CREB, p-CREB/CREB, and SYP as well as the average optical density of BrdU/NeuN double-labelled positive cells. Our study shows that lipopolysaccharides induce inflammation and nerve damage in hippocampal neurons, which are closely related to the pathological mechanism of depression. Xiaoyao Pills (XYW) play an important neuroprotective effect, which is related to its inhibition of neuronal inflammation and promoting the recovery of nerve injury. These results provide a pharmacologic basis for the treatment of depression of XYW in clinical application.

Inhibitory Effect of Jing-Fang Powder n-Butanol Extract and Its Isolated Fraction D on Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells.

The Jing-Fang powder n-butanol extract (JFNE) has anti-inflammatory properties; however, its active ingredient remains unknown. In addition, the mechanism by which JFNE exerts its anti-inflammatory effects on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells is yet to be explored. In this study, JFNE was isolated by chromatography to obtain fraction D. We found that pretreatment of LPS-induced RAW264.7 cells with JFNE and fraction D for 3 hours significantly reduced the levels of nitric oxide (NO), interleukin (IL)-1ß, and tumor necrosis factor-α (TNF-α) in the supernatant of cell cultures, and fraction D could also reduce the level of IL-6. In addition, JFNE and fraction D significantly reduced the mRNA expression of inducible NO synthase (iNOS), IL-6, IL-1ß, and TNF-α JFNE and fraction D significantly inhibited the phosphorylation of proteins and mRNA expression levels of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/AKT). Moreover, JFNE and fraction D significantly decreased the mRNA expression of iNOS, v-rel reticuloendotheliosis viral oncogene homolog A (RELA), and nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB1), whereas an increase in the mRNA expression of conserved helix-loop-helix ubiquitous kinase (CHUK) was observed. In addition, JFNE and fraction D downregulated the protein expression of iNOS, nuclear factor-κB (NF-κB) (p50), and phosphorylated NF-κB (p65). These results show that JFNE and its isolated fraction D exert specific anti-inflammation properties in LPS-stimulated RAW264.7 cells that are regulated by inhibition of the PI3K/Akt and NF-κB signaling pathways.

[Effects of Jingfang n-butanol extraction isolated fraction A on LPS-induced inflammation in RAW264.7 cells].

The LPS-induced RAW264. 7 cells inflammation model was used as a carrier to investigate the in vitro anti-inflammation effects of Jingfang n-butanol extraction(JFNE) isolated fraction A and explore its preliminary anti-inflammation mechanism by observing the regulatory effect on PI3 K/AKT signaling pathway and NF-κB pathway. The RAW264. 7 cells inflammation model was established by stimulating with LPS for 12 h. After 3 h pre-treatment with fraction A,the contents of interleukin-6(IL-6),interleukin-1ß(IL-1ß) and tumor necrosis factor(TNF-α) in the supernatant of RAW264. 7 cells inflammation model were determined by ELISA and the contents of NO in supernatant were assayed by Griess. Reverse transcription-polymerase chain reaction(RT-PCR) method was used to determine the expression of IL-6,IL-1ß,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammatory cells,and the expression levels of phosphorylated and total PI3 K/AKT protein,NF-κB p50,p65,p-p65,p105 protein in cells were determined via Western blot. In addition,LC-MS and database were used to identify the possible chemical constituents in fraction A. The results showed that fraction A could significantly reduce the release levels of NO,IL-6,IL-1ß and TNF-α in the supernatant and the expression of IL-6,IL-1ß,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammation model cells(P<0. 05 or P<0. 01) and significantly inhibit the phosphorylation expression levels of PI3 K and AKT protein and mRNA expressions(P<0. 05 or P<0. 01). Moreover,fraction A could significantly reduce the levels of NF-κB p50,p-p65 and i NOS protein,as well as NF-κB1,Rela mRNA expressions in RAW264. 7 cells,and increase the expression of CHUK gene.A total of 196 compounds were identified from fraction A in the composition analysis,and isoobtusilactone,5-O-methyl-vismitol,emebel(embelin) and prim-O-glucosylcimifugin showed high contents. The results all above showed that fraction A had a certain antiinflammatory effect in LPS-induced RAW264. 7 inflammation model cells,and its anti-inflammatory effects may be related to its regulatory effect on the activation of PI3 K/AKT signaling pathway and NF-kappa B signaling pathway. In addition,emblin may be its effective anti-inflammation chemical composition.

[Protective effect and mechanism of Xiaoyao San on lipopolysaccharide-induced hippocampal neurons injury].

To investigate the relationship between anti-depressant effect and hippocampal nerve growth of Xiaoyao San,the inflammatory model of hippocampal neuron was induced by lipopolysaccharide( LPS). The effect of Xiaoyao San serum( final concentration of4%,8%) on the cell proliferation activity was detected by immunofluorescence,the levels of BDNF and ß-NGF in the supernatant of hippocampal neurons were detected by ELISA,and the expressions of BDNF,NGF,Trk B,Trk A and CREB mRNA in cell lysate of hippocampal neuron were detected by PCR. Western blot was used to detect the expressions of Trk B,CREB,p-CREB and SYP protein in cell lysate of hippocampal neuron,and to reveal the neuroprotective effect and mechanism of Xiaoyao San. The results showed that8% Xiaoyao San serum could significantly increase in Brdu/Neu N ratio( P<0. 01). 4%,8% Xiaoyao San serum could significantly improve the levels of BDNF and ß-NGF in supernatant( P<0. 05 or P<0. 01),up-regulate the expression of BDNF,NGF,Trk B,Trk A,CREB mRNA and Trk B,p-CREB,SYP protein in cell lysate( P< 0. 05 or P< 0. 01). 8% Xiaoyao San serum could significantly increase CREB protein in cell lysate( P<0. 05),and elevate in p-CREB/CREB ratio( P<0. 01). All the above results indicate that Xiaoyao San has a certain protective effect on LPS induced hippocampal neuron injury,which suggests that the protective effect of Xiaoyao San is related to the promotion of hippocampal nerve growth,which is one of its antidepressant mechanisms.