Diterpenoid alkaloids isolated from Delphinium trichophorum alleviate pulmonary fibrosis via the TGF-ß/Smad pathway in 3T6 and HFL-1 cells.

Delphinium trichophorum Franch (DTF), a species endemic to China, has been widely used for centuries in Tibet as an indigenous medicine for treating cough, pneumonia, and pulmonary fibrosis. Hetisine-type C20-diterpenoid alkaloids have been reported to be characteristic and active ingredients. Herein, five ones with relatively high contents in D. trichophorum, including 2α,11α,13ß-triacetylhetisine (DTF1), trichodelphinine A (DTF2), trichodelphinine D (DTF3), 2α-acetyl-11α,13ß-dihydroxyhetisine (DTF4), and trichodelphinine C (DTF5), were investigated for anti-fibrosis effects using fibroblasts induced by TGF-ß1 or LPS for the first time. The results showed that all five tested compounds decreased hydroxyproline (HYP) levels and inhibited the abnormal proliferation of 3T6 and HFL-1 cells induced by either TGF-ß1 or LPS. Moreover, DTF1 and DTF2 attenuated the production of collagen (Col-1 and Col-3) at relatively low doses, suggesting their higher efficiency among the five alkaloids. Based on large-scale ligand-based pharmacophore modeling, TGFBR1 was screened as a potential target for these tested alkaloids. The molecular docking results also exhibited high-affinity interactions between TGFBR1 and five alkaloids, especially DTF1 and DTF2. Further experiments revealed that DTF1 and DTF2 could inhibit the expression of TGF-ß1 and α-SMA and the phosphorylation of Smad3 and Smad4 while restoring the expression of Smad7 protein. Overall, DTF1 and DTF2 may reduce collagen generation and delay the development of pulmonary fibrosis by inhibiting the activation of the TGF-ß/Smad signaling pathway. Our results provide experimental and theoretical evidence for DTF1 and DTF2 as superior candidates for further development of anti-fibrotic drugs.

Trial of transcutaneous electrical acupoint stimulation in laryngopharyngeal reflux disease: study protocol for a randomized controlled trial.

BACKGROUND: Patients with persistent globus sensation, throat clearing, chronic cough, hoarseness, and other throat symptoms account for a large proportion of patients in ears, nose, and throat clinics. Laryngopharyngeal reflux disease (LPRD) is increasingly valued by otolaryngologists. Transcutaneous electrical acupoint stimulation (TEAS) is possibly a new method for the treatment of LPRD. This trial aims to determine whether TEAS combined with proton pump inhibitor (PPI) is better than PPI alone in the treatment of LPRD. METHODS: This prospective randomized controlled trial will be implemented in a tertiary hospital in China. Seventy patients diagnosed with LPRD will be randomly assigned to the TEAS + PPI group (intervention group) or PPI group (control group), at a ratio of 1:1. In addition to using PPI, the intervention group will receive TEAS at four groups of acupoints, and each group will be treated for 15 min, once for 60 min, five times a week, for 12 weeks, 60 times. The main outcome will be changes in the Reflux Symptom Index scores at 4, 12, and 24 weeks after treatment. The secondary outcomes will include changes in the reflux finding score, Laryngopharyngeal Reflux-Health-related Quality of Life score, and throat pain visual analog scale score. DISCUSSION: This trial will explore the feasibility of TEAS combined with PPI for the treatment of LPRD and provide potential evidence for its effectiveness and safety. The results of this study will be published in a peer-reviewed journal. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100046755 . Registered on May 28, 2021.

Diterpenoid Alkaloids Isolated from Delphinium brunonianum and Their Inhibitory Effects on Hepatocytes Lipid Accumulation.

This research aimed to excavate compounds with activity reducing hepatocytes lipid accumulation from Delphinium brunonianum. Four novel diterpenoid alkaloids, brunodelphinine B-E, were isolated from D. brunonianum together with eleven known diterpenoid alkaloids through a phytochemical investigation. Their structures were elucidated by comprehensive spectroscopy methods including HR-ESI-MS, NMR, IR, UV, CD, and single-crystal X-ray diffraction analysis. The inhibitory effects of a total of 15 diterpenoid alkaloids on hepatocytes lipid accumulation were evaluated using 0.5 mM FFA (oleate/palmitate 2:1 ratio) to induce buffalo rat liver (BRL) cells by measuring the levels of triglyceride (TG), total cholesterol (TC), alanine transaminase (ALT), aspartate transaminase (AST), and the staining of oil red O. The results show that five diterpenoid alkaloids-brunodelphinine E (4), delbruline (5), lycoctonine (7), delbrunine (8), and sharwuphinine A (12)-exhibited significant inhibitory effects on lipid accumulation in a dose-dependent manner and without cytotoxicity. Among them, sharwuphinine A (12) displayed the strongest inhibition of hepatocytes lipid accumulation in vitro. Our research increased the understanding on the chemical composition of D. brunonianum and provided experimental and theoretical evidence for the active ingredients screened from this herbal medicine in the treatment of the diseases related to lipid accumulation, such as non-alcoholic fatty liver disease and hyperlipidemia.

Authentication and Quality Assessment of Plumeriae Rubrae Flos by UHPLC-QTOF-MS/MS and UHPLC-DAD Combined With Chemometrics.

BACKGROUND: Plumeriae rubrae flos, the dried flowers of Plumeria rubra cv. acutifolia (PRCA), is one of the most important materials of herbal tea in China. Recently, due to the lack of effective quality evaluation standards, it has been found that Plumeria rubra (PR) and Plumeria rubra var. alba (PRVA) were pretended to be PRCA in herbal material markets. OBJECTIVE: To establish an effective method for comprehensive quality assessment on plumeriae rubrae flos, and distinguishing PRCA from its common adulterants, PR and PRVA. METHOD: In this study, a method combined application of ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), UHPLC with a diode array detector (UHPLC-DAD), and chemometrics was developed for qualitative and quantitative analysis of PRCA, PR, and PRVA, based on their multiple components. RESULTS: A total of 26 components were identified by UHPLC-QTOF-MS/MS, including nine flavonoids, eight iridoids, seven phenolic acids, and two coumarins from PRCA. Quantified fingerprints were established and validated using UHPLC-DAD based on 18 chemical markers in PRCA, PR, and PRVA samples. The multivariate statistical analysis of quantitative results demonstrated clear discrimination of PRCA, PR, and PRVA, which indicated that isoquercetin, luteolin-3'-O-ß-D-glucoside, 15-demethylplumieride P-E-coumarate, and 4-O-beta-glucopyranosyl-cis-coumaric acid could be considered the most obvious characteristic components for distinguishing PRCA from PR and PRVA. CONCLUSIONS: A combination of quantitative fingerprint and chemometrics analysis provided an effective and reliable strategy for the quality control of PRCA. HIGHLIGHTS: The current study was prospected to apply a comprehensive quality control method for plumeriae rubrae flos.

Pheophorbide co-encapsulated with Cisplatin in folate-decorated PLGA nanoparticles to treat nasopharyngeal carcinoma: Combination of chemotherapy and photodynamic therapy.

The adverse effect and drug resistance of Cisplatin (CDDP) could be potential reduced by delivering in targeted nanoparticles and by combining with adjuvant therapy such as photodynamic therapy. In this study, F/CDPR-NP was formulated and characterized for all the physicochemical, biological and in vivo analysis. The results obtained from various in vitro and biological studies showed that encapsulation of CDDP and PBR in PLGA nanoparticles results in controlled release of encapsulated drugs and exhibited significantly low cell viability in CNE-1 and HNE-1 cancer cells. F/CDPR-NP significantly prolonged the blood circulation of the encapsulated drugs. The AUC of CDDP from F/CDPR-NP (4-fold) was significantly higher compared to that of free CDDP and similarly significantly higher t1/2 for CDDP from F/CDPR-NP was observed. F/CDPR-NP in the presence of laser irradiation showed significant reduction in the tumor burden with low tumor cell proliferations compared to either CDPR-NP or free CDDP indicating the potential of targeted nanoparticles and photodynamic therapy. Overall, combination of treatment modalities and active targeting approach paved way for the higher antitumor activity in nasopharyngeal carcinoma model. The positive results from this study will show new horizon for the treatment of other cancer models.

A UPLC-MS/MS-based metabolomics analysis of the pharmacological mechanisms of rabdosia serra against cholestasis.

BACKGROUND: Rabdosia Serra, the dried aerial parts of Rabdosia serra (Maxim.) Hara (RS) from the Labiatae family, is a traditional Chinese herbal medicine called Xihuangcao. Although RS has been found to exert a therapeutic effect on cholestasis, the underlying molecular mechanism remains unclear. PURPOSE: This study was designed to investigate the pharmacological effect and mechanism of RS on cholestatic rats using metabolomics platform. METHODS: Histopathology and biochemical evaluations were performed to determine the therapeutic effect of RS and developed a rapid metabolite detection technology method based on UPLC-MS/MS to perform metabolomics research. Further, quantitative real-time polymerase chain reaction (RT-qPCR) was used to study the effect of RS on the bile acid metabolism pathway at the transcriptional level. RESULTS: RS significantly reduced the bile flow rates in cholestatic rats and decreased the levels of ALT, AST, TBA, T-BIL, and LDH, which were increased in the model group. Histological analysis showed that RS alleviated the liver injury induced by ANIT. Serum metabolomics results revealed 33 of the 37 biomarkers were found to be significantly altered by ANIT, and 26 were considerably changed following treatment with RS. Metabolic pathway analysis revealed four pathways such as primary bile acid biosynthesis, biosynthesis of unsaturated fatty acids, and arachidonic acid and tryptophan metabolism. The bile acid secretion process and the inflammation and oxidative stress processes are the major biochemical reactions following treatment with ANIT and RS. Bile acid-targeted metabolomics study showed that TCA, GCA, GCDCA, and GDCA might be sensitive biomarkers that induced liver injury. we found that treatment with RS regulated the levels of bile acid in the serum and liver and restored the proportion of bile acids, especially CA and conjugated bile acids, such as TCA and GCA, in the bile duct. RS increased the mRNA expression levels of FXR, SHP, BSEP, and MRP2 in livers, and IBABP, OST-α, and OST-ß in the ileum. CONCLUSION: In this study, RS was found to protect the liver by regulating multiple metabolic pathways and promoting the excretion of bile acids. Simultaneously, RS played an essential role in reversing the imbalance of bile acids and protected against cholestasis by regulating the expression of transporters associated with bile acids. We demonstrated the correlation between molecular mechanisms and metabolites, provide a reference for the fabrication of extracts that can be used to treat cholestasis.

MORIN MITIGATES OXIDATIVE STRESS, APOPTOSIS AND INFLAMMATION IN CEREBRAL ISCHEMIC RATS.

BACKGROUND: Morin is a flavanoid which exhibits potent antioxidant activity in various oxidative stress related diseases. The current study was attempted to scrutinize the preclinical bio-efficacy of morin on focal ischemia. METHODS: The animal model of focal cerebral ischemic injury was done by midbrain carotid artery occlusion (MCAO) method, followed by Morin (30mg/kg) administration for seven days. RESULTS: The outcome of the study showed that treatment with morin displayed positive effects in reducing the focal cerebral ischemia. This effect was evident with the improvements in neurological deficits, reduction in MDA content and elevation of antioxidant levels (SOD, GSH and Gpx). Furthermore, protein expression of Bax and caspase-3 were effectively down-regulated, whilst the expression of Bcl-2 was significantly elevated. On the other hand, the mRNA expression of proinflammatory cytokines was significantly reduced in focal cerebral ischemic rats upon morin intervention. CONCLUSION: Thus, the beneficial effects of morin on cerebral ischemia assault may result from the reduction of oxidative stress, inhibition of apoptosis and inflammation. The neuroprotective effects of morin supplement may serve as potent adjuvant in the amelioration of ischemic stroke.

The Inhibitory Effects of RFamide-Related Peptide 3 on Luteinizing Hormone Release Involves an Estradiol-Dependent Manner in Prepubertal but Not in Adult Female Mice.

The mammalian gonadotropin-inhibitory hormone (GnIH) ortholog, RFamide-related peptide (RFRP), is considered to act on gonadotropin-releasing hormone (GnRH) neurons and the pituitary to inhibit gonadotropin synthesis and release. However, there is little evidence documenting whether RFamide-related peptide 3 (RFRP-3) plays a primary role in inhibition of the hypothalamo-pituitary-gonadal (HPG) axis prior to the onset of puberty. The present study aimed to understand the functional significance of the neuropeptide on pubertal development. The developmental changes in reproductive-related gene expression at the mRNA level were investigated in the hypothalamus of female mice. The results indicated that RFRP-3 may be an endogenous inhibitory factor for the activation of the HPG axis prior to the onset of puberty. In addition, centrally administered RFRP-3 significantly suppressed plasma luteinizing hormone (LH) levels in prepubertal female mice. Surprisingly, centrally administered RFRP-3 had no effects on plasma LH levels in ovariectomized (OVX) prepubescent female mice. In contrast, RFRP-3 also inhibited plasma LH levels in OVX prepubescent female mice that were treated with 17beta-estradiol replacement. Our study also examined the effects of RFRP-3 on plasma LH release in adult female mice that were ovariectomized at dioestrus, with or without estradiol (E2). Our results showed that the inhibitory effects of RFRP-3 were independent of E2 status. Quantitative real-time PCR and immunohistochemistry analyses showed that RFRP-3 inhibited GnRH expression at both the mRNA and protein levels in the hypothalamus. These data demonstrated that RFRP-3 could effectively suppress pituitary LH release, via the inhibition of GnRH transcription and translation in prepubescent female mice, which is associated with estrogen signaling pathway and developmental stages.

One-pot enantioselective synthesis of functionalized pyranocoumarins and 2-amino-4H-chromenes: discovery of a type of potent antibacterial agent.

Function-oriented design and synthesis of chiral small molecules with novel activity is a key goal in modern organic chemistry. As multiple antibiotic-resistant pathogens are emerging and causing serious diseases, the need for practical routes for the development of new types of antibacterial agents is very urgent. Herein, we present a highly efficient process for the synthesis of optically active pyranocoumarins and 2-amino-4H-chromenes through an organocatalytic Knoevenagel/Michael/cyclization sequence, and the preliminary biological studies of these new heterocyclic compounds revealed potent antibacterial activity. This study provides a novel strategy for further research and development of new types of antibacterial agents effective against human pathogens.