Kaempferol ameliorates pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension rats via regulating Akt-GSK3ß-cyclin axis.

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is considered a major contributor to elevated pulmonary vascular resistance and a key mechanism of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Kaempferol is a natural flavonoid compound and can be derived from numerous common medicinal herbs and vegetables, which exhibit antiproliferative and proapoptotic properties, however, the effects of kaempferol on vascular remodeling in HPH remain unexplored. In this study, SD rats were placed in a hypobaric hypoxia chamber for four weeks to establish a pulmonary hypertension model and given either kaempferol or sildenafil (an inhibitor of PDE-5) during days 1-28, after which the hemodynamic parameter and pulmonary vascular morphometry were assessed. Furthermore, primary rat PASMCs were exposed to hypoxic conditions to generate a cell proliferation model, then incubated with either kaempferol or LY294002 (an inhibitor of PI3K). Immunoblotting and real-time quantitative PCR assessed the protein and mRNA expression levels in HPH rat lungs and PASMCs. We found that kaempferol reduced pulmonary artery pressure and pulmonary vascular remodeling, and alleviated right ventricular hypertrophy in HPH rats. The mechanistic analysis demonstrated that kaempferol reduced the protein levels of phosphorylation of Akt and GSK3ß, leading to decreased expression of pro-proliferation (CDK2, CDK4, Cyclin D1, and PCNA) and anti-apoptotic related proteins (Bcl-2) and increased expression of pro-apoptosis proteins (Bax and cleaved caspase 3). These results collectively demonstrate that kaempferol ameliorates HPH in rats by inhibiting PASMC proliferation and pro-apoptosis via modulation of the Akt/GSK3ß/CyclinD axis.

Computational method for rapid screening of the metabolites of Pulsatilla chinensis in rats using UHPLC-Q-TOF/MS combined with mass spectrum-based orthogonal projection.

Screening metabolites in vivo can be challenging due to the complexity of traditional Chinese medicine (TCM) and the ambiguous intracorporal process. To resolve this problem, we established the mass spectrum-based orthogonal projection (MSOP) method to differentiate prototype compounds from metabolites in vivo and applied it to the study of metabolites of Pulsatilla chinensis (PC). Initially, the validity and feasibility of the MSOP method were verified by using the ultra- high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) data of reference solution. Then, the MSOP method was applied to screen the metabolites of PC. A total of 63 metabolites were identified in vivo (urine, feces, bile, and plasma samples) and in vitro (intestinal bacteria biological sample). The results indicated that the main metabolic pathways of pentacyclic triterpenoids were demethylation, oxidation, dehydration, sulfation, and glucuronidation reactions. This study contributes to developing an integrated strategy based on chemometrics to characterize and classify the metabolism feature of pentacyclic triterpenoids of PC. This will support the scientific and rational application of PC in the clinic. The MSOP method based on the orthogonality of MS signals was used to differentiate the prototype compounds from metabolites in vivo. The method provides scientific and reliable support for fully understanding the metabolic fate of TCM.

Development of trisiloxane surfactant vesicles ultrasonic extraction method combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for the rapid differentiation of Bupleuri Radix based on metabolomics.

INTRODUCTION: Due to the variety, chemical composition and complex structure, the quality control of Bupleuri Radix (BR) is a challenging task. There are still many trace compounds in BR that are difficult to extract and detect. OBJECTIVE: To develop an innovative method of trisiloxane surfactant vesicles ultrasonic extraction (TSVUE) combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for the identification from Bupleurum chinense DC. (BC) to Bupleurum scorzonerifolium Willd (BS) based on metabolomics. METHODS: Based on extraction effect for BR, five different types of surfactants vesicles were prepared and compared. Then, a single-factor test and a response surface methodology study were adopted to obtain the optimal conditions for the surfactant vesicles ultrasonic extraction method. Finally, a non-targeted metabolomics method with information dependent acquisition mode was performed to analyse differential metabolites in BC and BS. RESULTS: Sugar-based surfactant containing trisiloxane [N-3-propyl-methyltrisiloxane-N-glucoheptonamne (Si(3)N-GHA)] displayed higher extraction efficiency compared to other types of surfactants when it comes to being used in pretreatment methods. And a TSVUE method was established and optimised. In total, 131 constituents were identified in two BR herbs, of which 35 were unreported, and 11 were characterised as chemical markers. CONCLUSIONS: This method provides promising perspectives for rapidly identifying trace compounds in complex systems of traditional Chinese medicine (TCM), as well as for laying the foundation in the identification of similar herbs from the same species. Meanwhile, these findings serve as a promising application of trisiloxane surfactant vesicles in the extraction field of TCM.

Anti-apoptotic effect of HeidihuangWan in renal tubular epithelial cells via PI3K/Akt/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Heidihuang Wan (HDHW) is a classic Chinese herbal formula, which was first recorded in the "Suwen Bingji Qiyi Baoming Collection" written by Liu Wansu during the Jin Dynasty (1115-1234 AD). It is commonly used clinically for the treatment of kidney diseases and its curative effect is stable. Previous animal experiments have confirmed that HDHW can effectively improve renal fibrosis. However, the underlying pharmacological mechanism remains unclear. AIMS OF THIS STUDY: Renal tubular epithelial cell (RTEC) apoptosis is one of the main pathological features of renal fibrosis. This study aimed to observe the effect and underlying mechanism of HDHW on the apoptosis of RTECs to further explore the pathological mechanism of HDHW against renal fibrosis. MATERIALS AND METHODS: We examined the HDHW composition in rat serum. In vitro, we first screened out the optimal intervention concentration of HDHW on RTECs using the MTT assay. Hypoxia/reoxygenation was then used to induce apoptosis of RTECs (H/R-RTECs), which were divided into H/R-RTEC, astragaloside IV (positive control), HDHW, and RTECs groups. After 48 h of drug intervention, apoptosis of RTECs was detected using flow cytometry and protein expression was detected by western blotting. The 5/6 nephrectomy rat model was constructed and divided into the normal control, 5/6 nephrectomy, HDHW, and astragaloside IV groups. After 8 weeks of treatment, TUNEL staining was used to detect cell apoptosis, and western blotting was used to detect protein expression. RESULTS: HDHW downregulated the expression of pro-apoptotic proteins Bax and Caspase3, up-regulated the expression of anti-apoptotic protein Bcl-2, activated the PI3K/Akt/mTOR signaling pathway, and reversed the early apoptosis of RTECs, thereby resisting the apoptosis of RTECs. CONCLUSION: HDHW inhibits apoptosis of RTECs by modulating the PI3K/Akt/mTOR signaling pathway. This study provides experimental evidence for the anti-fibrotic effect of HDHW on the kidneys and partially elucidates its pharmacological mechanism of action.

Srolo Bzhtang reduces inflammation and vascular remodeling via suppression of the MAPK/NF-κB signaling pathway in rats with pulmonary arterial hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Srolo Bzhtang (SBT), which consists of Solms-laubachia eurycarpa, Bergenia purpurascens, Glycyrrhiza uralensis, and lac secreted by Laccifer lacca Kerr (Lacciferidae Cockerell), is a well-known traditional Tibetan medicinal formula and was documented to cure "lung-heat" syndrome by eliminating "chiba" in the ancient Tibetan medical work Four Medical Tantras (Rgyud bzhi). Clinically, it is a therapy for pulmonary inflammatory disorders, such as pneumonia, chronic bronchitis, and chronic obstructive pulmonary disease. However, whether and how SBT participates in pulmonary arterial hypertension (PAH) is still unclear. AIM OF THE STUDY: We aimed to determine the role of SBT in attenuating pulmonary arterial pressure and vascular remodeling caused by monocrotaline (MCT) and hypoxia. To elucidate the potential mechanism underlying SBT-mediated PAH, we investigated the changes in inflammatory cytokines and mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) signaling pathway. MATERIALS AND METHODS: MCT- and hypoxia-induced PAH rat models were used. After administering SBT for four weeks, the rats were tested for hemodynamic indicators, hematological changes, pulmonary arterial morphological changes, and the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in serum and lung tissues. Protein expression of the MAPK/NF-κB signaling pathway was determined using western blotting. RESULTS: SBT reduced pulmonary arterial pressure, vascular remodeling, and the levels of inflammatory cytokines induced by MCT and hypoxia in rats. Furthermore, SBT significantly suppressed the MAPK/NF-κB signaling pathway. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that SBT alleviates MCT- and hypoxia-induced PAH in rats, which is related to its anti-inflammatory actions involving inhibition of the MAPK/NF-κB signaling pathway.

Luteolin ameliorates hypoxia-induced pulmonary hypertension via regulating HIF-2α-Arg-NO axis and PI3K-AKT-eNOS-NO signaling pathway.

BACKGROUND: Pulmonary hypertension (PH) is a devastating disease with poor prognosis and high mortality. Hypoxia induced pulmonary hypertension (HPH) is a persistent threat to human health, especially to people who live on high altitude plateau. Pulmonary vascular endothelial cell is involved in numerous pathophysiological processes, including in vasoconstriction, oxidative stress, cell growth and differentiation. Endothelial cells (ECs) are the first layer to be exposed to changed oxygen levels and hypoxia could lead to ECs dysfunction. Endothelial-derived nitric oxide (NO) is the most important bioactive molecule, which could regulate endothelial homeostasis. PH pathophysiology has been linked to the disruption of NO pathways. PURPOSE: Luteolin is a kind of plant active ingredient with multiple pharmacological activities. The purpose of this study is to detect the effect of luteolin on HPH with in vivo, ex vivo and in vitro analyses and to further elucidate luteolin's pharmaceutical mechanism with NO related signaling pathway regulation. METHODS: Hypobaric chamber was used to establish HPH animal model. Rats were intragastrically administrated luteolin for 28 days. Then hemodynamic indexes, histopathological changes, pulmonary artery endothelial function, NO content and arginase activity in lung tissue, NO related pathway proteins expression were measured to evaluate the effect of luteolin on HPH. PAECs were treated with 1% O2 and incubated with or without luteolin. PAECs vitality, NO content in cells supernatant, and NO related pathway proteins expression were tested to reveal the protective mechanism of luteolin. RESULTS: Luteolin decreased mean pulmonary hypertension of HPH rats, alleviated right ventricular and pulmonary vascular remodeling. Immunofluorescence staining (vWF), isolated perfused/ventilated rat lung experiment indicated that luteolin protected pulmonary vascular endothelial function of HPH rats. Luteolin increased NO content in PAECs supernatant while decreased NO level in lung tissues of HPH rats. Further, it was demonstrated that luteolin inhibited HIF-2α-Arg axis in PAECs and HPH rats. PI3K-AKT-eNOS signaling pathway was upregulated in PAECs, but which was downregulated in lung tissues of HPH rats. Pharmacological effect of luteolin was equivalent or better than sildenafil. CONCLUSION: Luteolin ameliorated HPH in rats by protecting pulmonary vascular endothelial function via regulating HIF-2α-Arg-NO axis and PI3K-AKT-eNOS-NO signaling pathway. This study may provide a novel perspective and approach to alleviate the devastating disease of HPH.

Effects of Acupuncture at Neiguan in Neural Activity of Related Brain Regions: A Resting-State fMRI Study in Anxiety.

Background: Acupuncture of PC6 points has the effects of calming, tranquilizing, regulating qi, and relieving pain and has been clinically found to alleviate anxiety disorders. To explore the mechanism of improvement at the Neiguan point acupuncture in anxiety patients, we used fMRI to observe the changes in brain function in patients with immediate anxiety before and after acupuncture at the Neiguan point. Subjects and Methods: The experiment followed the principle of randomized, single-blind design. Twenty-four anxiety volunteers (14 males and 10 females, 20-35 years old) were divided randomly into two groups: a group of acupuncture at Neiguan and a group of acupuncture at non-acupoint. Functional magnetic resonance imaging (fMRI) was applied to measure brain activity pre- and post-acupuncture. The amplitude of low-frequency fluctuations (ALFF) and seed-based functional connectivity (FC) was used to analyze the activity and network of brain regions. Statistical analysis was done using SPSS 21.0 and REST 1.8 software. Results: ALFF results revealed that post-acupuncture at Neiguan increased the activity of the left parahippocampal gyrus, fusiform gyrus, and right superior temporal gyrus and decreased the activity of the right middle frontal gyrus, right precuneus, and cuneus. Post-acupuncture at non-acupoint led to a significant ALFF increase in the thalamus and middle frontal gyrus. The ALFF in the left middle frontal gyrus was decreased. Functional connectivity in several anterior default mode network (DMN) regions and vermis cerebelli at left parahippocampal/fusiform gyri was increased, and connectivity in bilateral superior temporal gyri was decreased. FC with posterior DMN regions decreased at the right middle frontal gyrus, right precuneus, and cuneus. Conclusion: Our study elucidates that acupuncture at Neiguan modulates anxiety by activating or deactivating these brain anxiety-related regions and provides potential explanations for the application of PC6 acupuncture in mental diseases.

Integrative analyses of widely targeted metabolomic profiling and derivatization-based LC-MS/MS reveals metabolic changes of Zingiberis Rhizoma and its processed products.

Zingiberis Rhizoma (ZR) has nutritional value and application potentiality, while Zingiberis Rhizoma Praeparatum (ZRP) and Carbonised Ginger (CG) are two main processed products of ZR based on different methods. Here, we performed a widely targeted metabolomics method with Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH) mode to analyze differential metabolites in ZR, ZRP and CG. Additionally, the chemical derivatization was applied to characterize different submetabolomes and improve the separation effect and MS response of metabolites. In total, 369 metabolites were identified and divided into 14 categories, 104 of which were differential metabolites. Our results suggest that carbohydrates, nucleotides, organic acids, vitamins, lipids, indoles, alkaloids, and terpenes contributed to a downward trend after processing, but the maximum content of flavanones, phenylpropanes and polyphenols appeared in ZRP, and that of alcohols appeared in CG. These findings serve as promising perspectives for developing functional food in ZR, ZRP and CG.

Effect of Modified Xianglian Pingwei Powder plus Glutathione and Levofloxacin Hydrochloride on Patients with Liver Cirrhosis and Positive Small Intestinal Bacterial Overgrowth.

Liver cirrhosis is a common chronic disease in China. The effect of modified Xianglian Pingwei powder plus Western medicine in the treatment of liver cirrhosis and positive small intestinal bacterial overgrowth is promising. Totally, 100 patients with liver cirrhosis and positive intestinal bacterial overgrowth in Cangzhou Central Hospital from February 2020 to February 2021 were enrolled and randomized via the random number table method at a ratio of 1 : 1 into the study group and control group. The control group received glutathione and levofloxacin hydrochloride, and the study group received Xianglian Pingwei powder plus glutathione and levofloxacin hydrochloride. The traditional Chinese medicine (TCM) syndrome scores, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) levels of the two groups were decreased after treatment with lower results in the study group. Xianglian Pingwei powder plus glutathione and levofloxacin hydrochloride was associated with a significantly lower positive rate of small intestine bacterial growth, serum endotoxin level, and peripheral blood toll-like receptor 2 (TLR2) and TRL4 levels versus glutathione and levofloxacin hydrochloride. The combined medication achieved a higher efficacy (90.00%) versus glutathione and levofloxacin hydrochloride (66.00%). The two groups experienced similar safety. Xianglian Pingwei powder plus glutathione and levofloxacin hydrochloride achieved significant benefits of clinical efficacy with a high safety profile in patients with liver cirrhosis versus glutathione and levofloxacin hydrochloride.

Mechanism of Traditional Tibetan Medicine Grubthobrildkr Alleviated Gastric Ulcer Induced by Acute Systemic Hypoxia in Rats.

Objective: This study was aimed at investigating the potential mechanism of Grubthobrildkr (GTB) on systemic hypoxia-induced gastric ulcers in rats and at detecting the chemical profile of GTB. Methods: Male Sprague-Dawley rats were separated into control, hypoxia, hypoxia+omeprazole, and hypoxia+GTBs (0.25, 0.5, and 1.0 g·kg-1·d-1) groups. Systemic hypoxia was created in a hypobaric chamber to simulate 5000 m high altitude by adjusting the inner pressure and oxygen content for 6 days. After that, the ulcer index, pH, and volume of gastric juice were assessed. The levels of endothelin-1 (ET-1), gastrin (GAS), motilin (MTL), phospholipase A2 (PLA2), and prostaglandin E2 (PGE2) were detected by ELISA. The expression level of hydrogen potassium ATPase (H+-K+-ATPase), cyclooxygenase-1 (COX-1), and cyclooxygenase-2 (COX-2) was tested by western blotting. Chemical profile of GTB was revealed by UHPLC-Q-exactive hybrid quadrupole-orbitrap mass (UHPLC-Q-Orbitrap MS). Results: GTB decreased the ulcer index in rats under hypoxia for six days, which was related to increased pH and volume of gastric juice, enhanced MTL and PGE2 levels, and decreased ET-1 and PLA2 levels of gastric mucosa. Furthermore, GTB decreased the level of H+-K+-ATPase and COX-2 while increased COX-1 levels in gastric mucosal tissue. 44 constituents were identified by UHPLC-Q-Orbitrap MS in GTB. Conclusion: GTB exerted a gastroprotective effect to alleviate gastric ulceration induced by acute systemic hypoxia in rats. The effect of GTB increasing the volume and pH of gastric juice in rats under acute systemic hypoxia could be regulated by gastrointestinal hormones, including MTL and ET-1. Mechanically, gastrointestinal protection of GTB was based on inhibition of the protons pumping H+-K+-ATPase and regulation of prostaglandin family in rats.

Abnormal functional connectivity of brain regions associated with fear network model in panic disorder.

Background: Patients with panic disorder (PD) have an abnormal function in brain regions related to fear network is well recognised. However, the traditional fear network model (FNM) which was based on animals' horrible behaviours has been found that it's not enough to explain the pathological mechanism of PD. This study aims to explore brain regions' abnormalities in the new advanced FNM, and estimate whether it can better explain PD.Methods: Magnetic resonance imaging resting-state scans were acquired in 40 patients with PD (35 drug-naïve and 5 drug-free) and 40 healthy controls (HCs). Twelve brain regions in the advanced FNM were chosen as regions of interest (ROIs) to examine the group difference in the ROIs and whole-brain resting-state functional connectivity (rsFC).Results: We found significantly increased thalamic rsFC with the insula, compared with HCs. And it was significantly correlated with HAMA-somatic score. We also found increased thalamic rsFC with occipital gyrus, temporal gyrus, and frontal gyrus when compared with HCs.Conclusions: Taken together, PD patients exhibit abnormal rsFC alterations within the advanced FNM, especially the increased rsFC within thalamus-insula loop, suggesting that excessive sensitivity to external information plays an important role in PD. The advanced FNM may provide a fuller explanation about PD.

Quality control of Zingiberis Rhizoma and its processed products by UHPLC-Q-TOF/MS-based non-targeted metabonomics combining with SIBDV method.

Zingiberis Rhizoma (ZR) is a homologous plant with pungent tastes and aromas, which has unique nutritional value and tremendous application potentiality. Zingiberis Rhizoma Praeparatum (ZRP) and Carbonised Ginger (CG) are processed products of ZR through different processing methods, and they are commonly used ingredients in food supplements. This study used ZR, ZRP and CG from different batches to further understand composition differences after processing. Additionally, we performed non-targeted metabolomics-based profiling of gingerols by ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) in combination with multivariate analysis and compounds identification. In which, we developed a comprehensive SWATH-IDA bi-directionally verified (SIBDV) method integrating the advantages of Sequential Windowed Acquisition of all Theoretical fragment ions (SWATHTM) and traditional information-dependent acquisition (IDA) mode for characterization of gingerols. Potential chemical markers were selected by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) of chemometrics methods. After that, the threshold variable importance in projection (VIP) value and P value were employed to screen the valuable MS features for discriminating ZR, ZRP and CG. In total, 59 gingerols in the different samples were structurally identified. Results allowed the selection of 33 gingerols, which are nominated as novel markers for materials authentication in ZR, ZRP and CG. The analysis of the study showed that the content of gingerols showed a downward trend after processing, but shogaols and gingerone compounds had an upward trend, resulting in differences in application and pharmacodynamic efficacy. These findings provide promising perspectives in the quality control of ZR, ZRP and CG, as well as for laying the foundation in food design and development.

α-Glucosidase inhibitory and anti-inflammatory activities of dammarane triterpenoids from the leaves of Cyclocarya paliurus.

Diabetes mellitus is caused by chronic inflammation and affects millions of people worldwide. Cyclocarya paliurus leaves have been widely used in traditional folk tea as a remedy for diabetes, but the antidiabetic constituents remain to be further studied. The α-glucosidase inhibitory and anti-inflammatory activities were examined to evaluate their effects on diabetes mellitus, and bioassay-guided separation of C. paliurus leaves led to the identification of twenty dammarane saponins, including eleven new dammarane saponins (1-11). The structures of the isolates were elucidated by spectroscopic methods. Bioactivity assay results showed that compounds 1 and 2 strongly inhibited α-glucosidase activity, with IC50 values ranging from 257.74 µM, 282.23 µM, and strongly inhibited the release of NO, with IC50 values of 9.10 µM, 9.02 µM. Moreover, compound 2 significantly downregulated the mRNA expression of iNOS, COX-2, IL-1ß, NF-κB, IL-6 and TNF-α in LPS-mediated RAW 264.7 cells and markedly suppressed the protein expression of iNOS, NF-κB/p65, and COX-2. Dammarane glucoside 2 exhibited the strongest α-glucosidase inhibitory and anti-inflammatory activities. In addition, the structure-activity relationships (SARs) of the dammarane saponins were investigated. In summary, C. paliurus leaves showed marked α-glucosidase inhibitory and anti-inflammatory activities, and dammarane saponins are responsible for regulating α-glucosidase, inflammatory mediators, and mRNA and the protein expression of proinflammatory cytokines, which could be meaningful for discovering new antidiabetic agents.

Miniaturized solid-phase extraction using a mesoporous molecular sieve SBA-15 as sorbent for the determination of triterpenoid saponins from Pulsatilla chinensis by ultrahigh-performance liquid chromatography-charged aerosol detection.

A simple, sensitive and efficient solid-phase extraction method, combined with ultrahigh-performance liquid chromatography-charged aerosol detection, was applied to the pre-concentration and determination of four triterpenoid saponins from Pulsatilla chinensis (P. chinensis) ultrasound extract samples. Mesoporous molecular sieve Santa Barbara Amorphous 15 (SBA-15) displayed higher selectivity compared to Mobil Composition of Matter 41 (MCM-41), NH2-SBA-15 and mesoporous carbon when it comes to being used in pretreatment methods. It was applied as an effective sorbent in the SPE for the enrichment of the target analytes. Additionally, several key experimental parameters including the kinds of sorbents, the amount of SBA-15, the elute pH and types of elution solvent were investigated in detail. Under the optimized conditions, the satisfactory linearity (r2 ≥ 0.9940) was acquired and the limits of detection reached 0.461-0.976 µg/mL for the target analytes. The recoveries ranged from 95.1%-103.2%. The experimental results showed that SBA-15 was a candidate material for the purification and concentration of target triterpenoid saponins from complex P. chinensis samples. The study provided theoretical support for the application of mesoporous materials in the field of drug separation and provided references for the extraction and determination of trace compounds in the complex systems of traditional Chinese medicine.

3,4-seco-Dammarane Triterpenoid Saponins with Anti-Inflammatory Activity Isolated from the Leaves of Cyclocarya paliurus.

Cyclocarya paliurus is commonly used for the prevention and treatment of hypertension, diabetes, and inflammation in South China. Although research on the anti-inflammatory effects of C. paliurus leaves has been reported, no active anti-inflammatory compounds have been identified. In the present study, RAW 264.7 cells were used to establish a bioactivity-guided identification model to verify the inhibitory effects of C. paliurus leaves on inflammation and identify the anti-inflammatory constituents. The active fraction was isolated to yield 18 dammarane triterpenoid saponins, including 11 new 3,4-seco-dammarane triterpenoid saponins (1-11), the structures of which were identified on the basis of analyses of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) and comparison with literature data. Compounds 7, 8, 10, and 11 showed strong inhibition on nitric oxide (NO) productions, with IC50 values ranging from 8.23 to 11.23 µM. These four compounds significantly decreased the secretion of tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), and interleukin 6 (IL-6) in lipopolysaccharide-activated RAW 264.7 cells. Furthermore, compound 7 decreased the expression of the proteins cyclooxygenase-2 (COX-2), inducible nitric-oxide synthase (iNOS), and nuclear factor kappa-B (NF-κB/p65). In addition, the structure-activity relationships of the isolates were investigated. The results suggest that 3,4-seco-dammarane triterpenoid saponins may be used as potential anti-inflammatory drugs and warrant further investigation.

Tsantan Sumtang attenuated chronic hypoxia-induced right ventricular structure remodeling and fibrosis by equilibrating local ACE-AngII-AT1R/ACE2-Ang1-7-Mas axis in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Tsantan Sumtang, which consists of Choerospondias axillaris (Roxb.) Burtt et Hill, Myristica fragrans Houtt and Santalum album L, is a traditional and common prescription of Tibetan medicine. Tsantan Sumtang originates from Four Tantra with properties of nourishing heart and has been used as a folk medicine for cardiovascular diseases and heart failure in Qinghai, Tibet and Inner Mongolia. Our previous studies found that Tsantan Sumtang showed beneficial effects on right ventricular structure in hypoxia rats, while the underling mechanism remains unclear. AIM OF THE STUDY: To elucidate the underlying mechanisms of Tsantan Sumtang attenuated right ventricular (RV) remodeling and fibrosis of chronic hypoxia-induced pulmonary arterial hypertension (HPAH) rats. MATERIALS AND METHODS: Fifty male Sprague Dawley (SD) rats (170 ± 20 g) were randomly divided into control group, hypoxia group, and hypoxia + Tsantan Sumtang groups (1.0 g·â€¯kg-1·day-1, 1.25 g·â€¯kg-1·day-1, 1.5 g ·kg-1·day-1). Rats in the hypoxia group and hypoxia + Tsantan Sumtang groups were maintained in a hypobaric chamber by adjusting the inner pressure and oxygen content to simulate an altitude of 4500 m for 28 days. The mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI), the ratio of RV weight to tibia length (TL) (RV/TL), heart rate (HR) and RV systolic pressure (RVSP) was determined. Histomorphological assay of RV structure was evaluated by hematoxylin and eosin (HE) staining. RV tissue fibrosis was assessed by collagen proportion area (CPA), collagen I, collagen III and hydroxyproline content. CPA was obtained by picro-sirius red staining (PSR). The expression of collagen I and collagen III were detected by immunohistochemistry and western blotting. The hydroxyproline content was detected by alkaline hydrolysis. In addition, the level of angiotensin II (AngII) and angiotensin 1-7 (Ang1-7) in RV tissue was tested by enzyme-linked immune sorbent assay (ELISA). Protein expression of angiotensin-converting enzyme (ACE), AngII, AngII type 1 receptor (AT1R), angiotensin-converting enzyme 2 (ACE2), Mas receptor (Mas) were determined by immunohistochemistry and western blotting. mRNA level of ACE, AT1R, ACE2, Mas were tested by qPCR. The chemical profile of Tsantan Sumtang was revealed by UHPLC-Q-Exactive hybrid quadrupole-orbitrap mass analysis. RESULTS: Our results showed that RVHI, RV/TL and RVSP were significantly increased in HPAH rat. Furthermore, levels of collagen I, collagen III and hydroxyproline were up-regulated in RV tissue under hypoxia. We found that RV hypertrophy and fibrosis were associated with increased expression of ACE, AngII, AT1R as well as decreased expression of ACE2, Ang1-7 and Mas. RV remodeling and fibrosis were attenuated after Tsantan Sumtang administration by up-regulating ACE2 and Mas level as well as down-regulating ACE, AngII and AT1R levels in RV tissue. 35 constituents in Tsantan Sumtang were identified. CONCLUSION: Tsantan Sumtang attenuated RV remodeling and fibrosis in rat exposed to chronic hypoxia. The pharmacological effect of Tsantan Sumtang was based on equilibrating ACE-AngII-AT1R and ACE2-Ang1-7-Mas axis of RV tissue in HPAH rat.

Srolo Bzhtang, a traditional Tibetan medicine formula, inhibits cigarette smoke induced airway inflammation and muc5ac hypersecretion via suppressing IL-13/STAT6 signaling pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Srolo Bzhtang (SBT), a traditional Tibetan medicine formula, was composed of three herbs, Solms-Laubachia eurycarpa, Bergenia purpurascens, Glycyrrhiza uralensis, and one lac, and was first documented in the ancient Tibetan medical work Four Medical Tantras (rGyud-bzhi) in the eighth century AD. It has been widely used to treat lung "phlegm-heat" syndromes such as chronic bronchitis and chronic obstructive pulmonary disease (COPD). OBJECTIVE: The aim of this study was to evaluate the potential influences of aqueous extract of SBT on airway inflammation and mucus secretion and to reveal the underlying mechanism in a rat model of cigarette smoke (CS)-induced chronic bronchitis (CB). MATERIALS AND METHODS: Sixty male Sprague-Dawley rats were randomly divided to six groups: control (room air exposure), model (CS exposure), DEX (CS exposure and 0.2 mg/kg/day dexamethasone), and three SBT (CS exposure and 1.67, 2.50, and 3.34 g/kg/day SBT) groups. DEX and the three doses of SBT were administered by oral gavage every day for eight weeks. Pathological changes and mucus expression in the lung tissue were determined by hematoxylin and eosin (H&E), Alcian blue-periodic acid-Schiff (AB-PAS) and immunohistochemical staining. The levels of cytokines in bronchoalveolar lavage fluid (BALF) were assessed by ELISA. Western blot analysis and qRT-PCR were performed to explore the effects of SBT on the expression of IL-13, STAT6 and MUC5AC. RESULTS: Pretreatment with SBT attenuated the TNF-α, IL-8, IL-13 expression levels in BALF and the inflammatory cell infiltration in bronchial walls and peribronchial lung tissue. SBT exhibited a dose-dependent downregulation of MUC5AC expression as assessed by AB-PAS and immunohistochemical staining. The protein and mRNA levels of IL-13, STAT6/p-STAT6 and MUC5AC were also downregulated by SBT preconditioning. CONCLUSION: These results for the first time demonstrated that SBT exhibited protective effects on CS-induced airway inflammation and MUC5AC hypersecretion, which might be related to the downregulation of the IL-13/STAT6 signaling pathway.

Two new C21 steroidal glycosides isolated from Cynanchum komarovii.

The present study was designed to further investigate the C21 steroidal glycosides in Cynanchum plants. Two new steroidal glycosides based on a 13, 14:14, 15-disecopregnane-type aglycone, komaroside P (1) and komaroside Q (2), together with three known compounds (3-5) were isolated from the whole herbs of Cynanchum komarovii. The aglycones of compounds 1 and 2 were two new disecopregnane. Their structures were elucidated on the basis of 1D, 2D NMR spectroscopic data and acid hydrolysis. All the compounds (1-5) showed potent inhibitory activities against human leukemia cell lines (HL-60) with IC50 values ranging from 16.6 to 26.3 µmol·L-1, compared to the positive control 5-fluorouracil (6.4 µmol·L-1).

Bioactive fraction of Rhodiola algida against chronic hypoxia-induced pulmonary arterial hypertension and its anti-proliferation mechanism in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola algida var. tangutica (Maxim.) S.H. Fu is a perennial plant of the Crassulaceae family that grows in the mountainous regions of Asia. The rhizome and roots of this plant have been long used as Tibetan folk medicine for preventing high latitude sickness. AIM OF THE STUDY: The aim of this study was to determine the effect of bioactive fraction from R. algida (ACRT) on chronic hypoxia-induced pulmonary arterial hypertension (HPAH) and to understand the possible mechanism of its pharmacodynamic actions. MATERIALS AND METHODS: Male Sprague-Dawley rats were separated into five groups: control group, hypoxia group, and hypoxia+ACRT groups (62.5, 125, and 250mg/kg/day of ACRT). The chronic hypoxic environment was created in a hypobaric chamber by adjusting the inner pressure and oxygen content for 4 weeks. After 4 weeks, major physiological parameters of pulmonary arterial hypertension such as mPAP, right ventricle index (RV/LV+S, RVHI), hematocrit (Hct) levels and the medial vessel thickness (wt%) were measured. Protein and mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, p27Kip1 and cyclin-dependent kinase 4 (CDK4)) were detected by western blotting and real time PCR respectively. Chemical profile of ACRT was revealed by ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS). RESULTS: The results showed that a successful HPAH rat model was established in a hypobaric chamber for 4 weeks, as indicated by the significant increase in mPAP, RV/LV+S, RV/BW and wt%. Compared with the normal group, administration of ACRT reduced mPAP, right ventricular hypertrophy, pulmonary small artery wall thickness, and damage in ultrastructure induced by hypoxia in rats. PCNA, cyclin D1, and CDK4 expression was reduced (p<0.05), and p27Kip1 expression increased (p<0.05) in hypoxia+ACRT groups compared to hypoxia. 38 constituents in bioactive fraction were identified by UHPLC-Q-TOF-MS/MS. CONCLUSION: Our results suggest that ACRT could alleviate chronic hypoxia-induced pulmonary arterial hypertension. And its anti-proliferation mechanism in rats based on decreasing PCNA, cyclin D1, CDK4 expression level and inhibiting p27Kip1 degradation.

Tsantan Sumtang Alleviates Chronic Hypoxia-Induced Pulmonary Hypertension by Inhibiting Proliferation of Pulmonary Vascular Cells.

Hypoxia-induced pulmonary hypertension (HPH) is a severe condition associated with significant morbidity and mortality in people living at high altitude. Tsantan Sumtang, a traditional Tibetan medicine, has been routinely used for the treatment of cardiopyretic disease, as well as stenocardia. Interestingly, our previous research found that Tsantan Sumtang improved HPH in rats maintaining in a hypobaric chamber. We performed a series of experiments to test the indexes of vasoconstriction and vascular remodeling, the key pathophysiological characteristics of HPH. Our results showed that Tsantan Sumtang relaxed noradrenaline (NE)-precontracted rat pulmonary artery rings in a concentration-dependent manner in vitro. The PGI2-cAMP (prostaglandin I2-cyclic adenosine monophosphate) pathway, NO-cGMP (nitric oxide-cyclic guanosine monophosphate) pathway, and the opening of K+ channels (inward rectifier K+ channels, large conductance Ca2+-activated K+ channels, and voltage-dependent K+ channels) might play major roles in the vasorelaxation effect. In vivo, the administration of Tsantan Sumtang resulted in a substantial decrease in the rat mean pulmonary artery pressure (mPAP) and the right ventricular hypertrophy index (RVHI). The reduction of thickness of small pulmonary arterial wall and the WT% (the ratio of the vascular wall thickness to the vascular diameter) were observed. The smooth muscle muscularization of the arterials was alleviated by Tsantan Sumtang treatment at the same time. Tsantan Sumtang also reduced remodeling of pulmonary arterioles by suppressing the expression of proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), cyclin D1, and cyclin-dependent kinase 4 (CDK4) through inhibition of p27Kip1 degradation. Therefore, Tsantan Sumtang could be applied as a preventative medication for HPH, which would be a new use for this traditional medicine.