Long-Circulating and Brain-Targeted Liposomes Loaded with Isoliquiritigenin: Formation, Characterization, Pharmacokinetics, and Distribution.

Isoliquiritigenin (ISL) has excellent neuroprotective effects. However, its limitations, including poor solubility, low bioavailability, and low accumulation in the brain, restrict its clinical promotion. In this study, a novel type of ISL-loaded liposome (ISL-LP) modified with the brain-targeting polypeptide angiopep-2 was prepared to improve these properties. The zeta potential, morphology, particle size, encapsulation efficiency, drug loading, and in vitro release of ISL-LP were evaluated. The pharmacokinetics and tissue distribution of ISL and ISL-LP were also investigated. The results demonstrated that ISL-LP had an average particle size of 89.36 ± 5.04 nm, a polymer dispersity index of 0.17 ± 0.03, a zeta potential of -20.27 ± 2.18 mV, and an encapsulation efficiency of 75.04 ± 3.28%. The in vitro release experiments indicate that ISL-LP is a desirable sustained-release system. After intravenous administration, LPC-LP prolonged the circulation time of ISL in vivo and enhanced its relative brain uptake. In conclusion, ISL-LP could serve as a promising brain-targeting system for the treatment and prevention of central nervous system (CNS) disorders.

Integrated Approaches Revealed the Therapeutic Mechanisms of Zuojin Pill Against Gastric Mucosa Injury in a Rat Model with Chronic Atrophic Gastritis.

Background: The Zuojin Pill (ZJP) is widely used for treating chronic atrophic gastritis (CAG) in clinical practice, effectively ameliorating symptoms such as vomiting, pain, and abdominal distension in patients. However, the underlying mechanisms of ZJP in treating CAG has not been fully elucidated. Purpose: This study aimed to clarify the characteristic function of ZJP in the treatment of CAG and its potential mechanism. Methods: The CAG model was established by alternant administrations of ammonia solution and sodium deoxycholate, as well as an irregular diet. Therapeutic effects of ZJP on body weight, serum biochemical indexes and general condition were analyzed. HE staining and AB-PAS staining were analyzed to characterize the mucosal injury and the thickness of gastric mucosa. Furthermore, network pharmacology and molecular docking were used to predict the regulatory mechanism and main active components of ZJP in CAG treatment. RT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to measure the expression levels of apoptosis-related proteins, gastric mucosal barrier-associated proteins and PI3K/Akt signaling pathway proteins. Results: The results demonstrated that ZJP significantly improved the general state of CAG rats, alleviated weight loss and gastric histological damage and reduced the serum biochemical indicators. Network pharmacology and molecular docking found that ZJP in treating CAG by inhibiting inflammation, suppressing apoptosis, and protecting the gastric mucosal barrier via the PI3K/Akt signaling pathway. Further experiments confirmed that ZJP obviously modulated the expression of key proteins involved in gastric mucosal cell apoptosis, such as Bax, Bad, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, Cytochrome C, Bcl-2, and Bcl-xl. Moreover, ZJP significantly reversed the protein expression of Occludin, ZO-1, Claudin-4 and E-cadherin. Conclusion: Our study revealed that ZJP treats CAG by inhibiting the PI3K/Akt signaling pathway. This research provided a scientific basis for the rational use of ZJP in clinical practice.

Multi-omic approaches provide insights into the molecular mechanisms of Sojae semen germinatum water extract against overactive bladder.

Sojae semen germinatum (SSG) is derived from mature soybean seeds that have been germinated and dried, typically with sprouts measuring approximately 0.5 cm in length. SSG is traditionally known for its properties in clearing heat and moisture. Nevertheless, limited information was reported on the effects and mechanisms of SSG in alleviating urinary symptoms. This study employed urodynamic parameters to investigate the therapeutic effect of SSG water extract on overactive bladder (OAB) in the rat model with benign prostatic hyperplasia. Through a combination of transcriptomic and metabolomic analyses, the pathways and key proteins of the SSG treatment for OAB were identified and validated by ELISA and Western blotting. Furthermore, network pharmacology elucidated the roles of SSG's isoflavones acting on the target which was identified by above-mentioned multi-omics analysis. Our results indicate that SSG water extract significantly mitigated OAB by down-regulating the PGE2/EP1/PLCß2/p-MLC signaling pathway. It was speculated that the active ingredient in the SSG on EP1 was genistein. This study provided valuable insights into the molecular mechanisms of SSG water extract, emphasizing the multi-target characteristics and critical pathways in improving OAB. Furthermore, this study contributes to the potential utilization of SSG as a functional food.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Long-Circulation and Brain Targeted Isoliquiritigenin Micelle Nanoparticles: Formation, Characterization, Tissue Distribution, Pharmacokinetics and Effects for Ischemic Stroke.

Purpose: We designed a novel isoliquiritigenin (ISL) loaded micelle prepared with DSPE-PEG2000 as the drug carrier modified with the brain-targeting polypeptide angiopep-2 to improve the poor water solubility and low bioavailability of ISL for the treatment of acute ischemic stroke. Methods: Thin film evaporation was used to synthesize the ISL micelles (ISL-M) modified with angiopep-2 as the brain targeted ligands. The morphology of the micelles was observed by the TEM. The particle size and zeta potential were measured via the nanometer particle size analyzer. The drug loading, encapsulation and in vitro release rates of micelles were detected by the HPLC. The UPLC-ESI-MS/MS methods were used to measure the ISL concentrations of ISL in plasma and main tissues after intravenous administration, and compared the pharmacokinetics and tissue distributions between ISL and ISL-M. In the MCAO mice model, the protective effects of ISL and ISL-M were confirmed via the behavioral and molecular biology experiments. Results: The results showed that the drug loading of ISL-M was 7.63 ± 2.62%, the encapsulation efficiency was 68.17 ± 6.23%, the particle size was 40.87 ± 4.82 nm, and the zeta potential was -34.23 ± 3.35 mV. The in vitro release experiments showed that ISL-M had good sustained-release effect and pH sensitivity. Compared with ISL monomers, the ISL-M could significantly prolong the in vivo circulation time of ISL and enhance the accumulation in the brain tissues. The ISL-M could ameliorate the brain injury induced by the MCAO mice via inhibition of cellular autophagy and neuronal apoptosis. There were no the cellular structural damages and other adverse effects for ISL-M on the main tissues and organs. Conclusion: The ISL-M could serve as a promising and ideal drug candidate for the clinical application of ISL in the treatment of acute ischemic stroke.

Scutellarin-induced A549 cell apoptosis depends on activation of the transforming growth factor-ß1/smad2/ROS/caspase-3 pathway.

Scutellarin plays an anti-tumor role in A549 lung cancer cells, but the underlying mechanism is unclear. In this study, scutellarin was used to treat A549 cells for 12, 24, and 48 h, followed by the addition of Tempo, a selective scavenger of mitochondrial reactive oxygen species (ROS) and SB431542, a transforming growth factor (TGF)-ß1 receptor inhibitor. A dihydroethidium fluorescence probe was used to measure the intracellular ROS level, Cell Counting Kit-8 (CCK-8) was used to detect cell viability, and flow cytometry was performed to examine apoptosis. Western blots were used to detect the total protein level of TGF-ß1, p-smad2, and cleaved caspase-3 in A549 cells. The results showed that scutellarin significantly inhibited cell viability and increased apoptosis. Scutellarin also promoted intracellular ROS production, TGF-ß1/smad2 signaling pathway activation, and cleaved caspase-3 expression, which was partly reversed by Tempo. Moreover, scutellarin-induced intracellular ROS production and cleaved caspase-3 expression were inhibited by blocking the TGF-ß1/smad2 pathway with SB431542. In conclusion, scutellarin promoted apoptosis and intracellular ROS accumulation, which could be abrogated by Tempo and SB431542 treatment in A549 cells. Our study indicated that scutellarin induced A549 cell apoptosis via the TGF-ß1/smad2/ROS/caspase-3 pathway.

The bioinformatics and metabolomics research on anti-hypoxic molecular mechanisms of Salidroside via regulating the PTEN mediated PI3K/Akt/NF-κB signaling pathway.

Salidroside (SAL), a major bioactive compound of Rhodiola crenulata, has significant anti-hypoxia effect, however, its underlying molecular mechanism has not been elucidated. In order to explore the protective mechanism of SAL, the lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide dismutase (SOD) and hypoxia-induced factor 1α (HIF-1α) were measured to establish the PC12 cell hypoxic model. Cell staining and cell viability analyses were performed to evaluate the protective effects of SAL. The metabolomics and bioinformatics methods were used to explore the protective effects of salidroside under hypoxia condition. The metabolite-protein interaction networks were further established and the protein expression level was examined by Western blotting. The results showed that 59 endogenous metabolites changed and the expression of the hub proteins of CK2, p-PTEN/PTEN, PI3K, p-Akt/Akt, NF-κB p65 and Bcl-2 were increased, suggesting that SAL could increase the expression of CK2, which induced the phosphorylation and inactivation of PTEN, reduced the inhibitory effect on PI3K signaling pathways and activated the PI3K/Akt/NF-κB survival signaling pathway. Our study provided an important insight to reveal the protective molecular mechanism of SAL as a novel drug candidate.

Development of long-circulating lapachol nanoparticles: formation, characterization, pharmacokinetics, distribution and cytotoxicity.

Lapachol is an active compound for the treatment of malignant brain glioma. However, its physicochemical properties limit its clinical application. The purpose of this study is to develop a nano-drug delivery system (LPC-LP) loaded with lapachol (LPC), which remarkably prolongs the half-life in the body, and increases the brain intake, therefore, achieving a better anticancer effect in the treatment of glioma. In order to optimize the formulation of liposomes, an orthogonal design was adopted with entrapment efficiency (EE) as the index. The characterization of the optimized formulation was evaluated in vitro. To assess the safety profile and effect of LPC-LP, a rapid and sensitive ultra-fast liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for studying the pharmacokinetics and brain distribution of LPC-LP and LPC. Finally, the cytotoxicity of the two preparations on C6 cells was studied by the MTT assay. The results showed that the average particle size of LPC-LP was 85.92 ± 2.35 nm, the EE of liposomes was 92.52 ± 1.81%, and the charge potential was -40.70 ± 9.20 mV. An in vitro release study showed that the release of lapachol from LPC-LP was delayed compared to LPC, indicating that LPC-LP was a sustained and controlled release system. The UPLC-MS/MS method was fully validated in both plasma and brain tissue according to the Food and Drug Administration (FDA) recommended guidelines, and successfully used for quantification of lapachol in vivo. After intravenous administration, LPC-LP prolonged circulation time of lapachol in the body and increased brain intake. Besides, the MTT results revealed that the IC50 value of LPC-LP on C6 cells significantly decreased, compared with LPC, which further confirmed that LPC-LP enhanced the inhibition of C6 cells and improved the anti-glioma effect. In conclusion, LPC-LP could serve as a promising candidate for the clinical application of lapachol in the treatment of glioma.

Network pharmacology exploration reveals the bioactive compounds and molecular mechanisms of Li-Ru-Kang against hyperplasia of mammary gland.

Li-Ru-Kang (LRK) has been commonly used in the treatment of hyperplasia of mammary gland (HMG) as a cipher prescription and achieved obvious therapeutic effects. However, the bioactive compounds and underlying pharmacological mechanisms remain unclear. This study aims to decipher the bioactive compounds and potential action mechanisms of LRK in the treatment of HMG using an integrated pharmacology approach. The ingredients of LRK and the corresponding drug targets were retrieved through drug target databases and were used to construct the "compound-target-disease" network and function-pathway network. Ultimately, 89 compounds and 2150 drug targets were collected. Gene ontology enrichment analysis revealed that mammary gland alveolus development and mammary gland lobule development were the key biological processes and were regulated simultaneously by three direct targets, including androgen receptor (AR), estrogen receptor (ER) and cyclin-D1. Moreover, 14 compounds of LRK were directly involved in the regulation of the three aforementioned targets. KEGG pathway enrichment analysis found that five signaling pathways and seven direct targets were closely related with HMG treatment by LRK. The results of animal experiments showed that LRK significantly improved the histopathological status of HMG in rats. Additionally, LRK markedly regulated the protein expressions of AR, cyclin-D1, MMP2, MMP3 and MMP9. But interestingly, the effect of LRK on ER was not obvious. This study demonstrated that LRK exerted its therapeutic efficacy based on multi-components, multi-targets and multi-pathways. This research confirms the advantages of network pharmacology analyses and the necessity for experimental verification.

Protective effects of higenamine combined with [6]-gingerol against doxorubicin-induced mitochondrial dysfunction and toxicity in H9c2 cells and potential mechanisms.

Higenamine (HG) is a well-known selective activator of beta2-adrenergic receptor (ß2-AR) with a positive inotropic effect. The present study showed that HG combined with [6]-gingerol (HG/[6]-GR) protects H9c2 cells from doxorubicin (DOX)-induced mitochondrial energy metabolism disorder and respiratory dysfunction. H9c2 cells were pretreated with HG/[6]-GR for 2 h before DOX treatment in all procedures. Cell viability was quantified by a cell counting kit­8 assay. Cardiomyocyte morphology, proliferation, and mitochondrial function were detected by a high content screening (HCS) assay. Cell mitochondrial stress was measured by a Seahorse XFp analyzer. To further investigate the protective mechanism of HG/[6]-GR, mRNA and protein expression levels of PPARα/PGC-1α/Sirt3 pathway-related molecules were detected. The present data demonstrated that protective effects of HG/[6]-GR combination were presented in mitochondria, which increased cell viability, ameliorated DOX-induced mitochondrial dysfunction, increased mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Most importantly, the protective effects were abrogated by GW6471 (a PPARα inhibitor) and ameliorated by Wy14643 (a PPARα agonist). Moreover, the combined use of HG and [6]-GR exerted more profound protective effects than either drug as a single agent. In conclusion, the results suggested that HG/[6]-GR ameliorates DOX-induced mitochondrial energy metabolism disorder and respiratory function impairment in H9c2 cells, and it indicated that the protective mechanism may be related to upregulation of the PPARα/PGC-1α/Sirt3 pathway, which promotes mitochondrial energy metabolism and protects against heart failure.

Phencynonate S-isomer as a eutomer is a novel central anticholinergic drug for anti-motion sickness.

To compare and evaluate the differences of stereoselective activity, the binding affinity, metabolism, transport and molecular docking of phencynonate isomers to muscarinic acetylcholine receptor (mAChR) were investigated in this study. The rotation stimulation and locomotor experiments were used to evaluate anti-motion sickness effects. The competitive affinity with [3H]-QNB and molecular docking were used for studying the interactions between the two isomers and mAChR. The stereoselective mechanism of isomers was investigated by incubation with rat liver microsomes, a protein binding assay and membrane permeability assay across a Caco-2 cell monolayer using a chiral column HPLC method. The results indicated that S-isomer was more effective against motion sickness and had not anxiogenic action at therapeutic doses. S-isomer has the higher affinity and activity for mAChR in cerebral cortex and acted as a competitive mAChR antagonist. The stereoselective elimination of S-isomer was primarily affected by CYP1B1 and 17A1 enzymes, resulting in a higher metabolic stability and slower elimination. Phencynonate S isomer, as a eutomer and central anticholinergic chiral drug, is a novel anti-motion sickness drug with higher efficacy and lower central side effect. Our data assisted the development of a novel drug and eventual use of S-isomer in clinical practice.

Anti-Inflammatory Effect of a TCM Formula Li-Ru-Kang in Rats With Hyperplasia of Mammary Gland and the Underlying Biological Mechanisms.

Li-Ru-Kang (LRK), a formula of eight traditional Chinese medicines (TCM), has been used to treat hyperplasia of mammary gland (HMG) in TCM clinics. However, how LRK works in HMG patients is unclear. To explore the possible mechanisms of LRK against HMG, the network pharmacology was used to screen the potential targets and possible pathways that involved in LRK treated HMG. Rat HMG model induced by estrogen and progesterone was used to further verify the effects of the key molecules of LRK selected from the enriched pathways on HMG. Nipple heights and diameters were measured and uterus index was calculated. The histopathological changes of mammary gland tissue were detected by hematoxylin-eosin (H&E) staining. Western blot was used to detect the phosphorylation of ERK, JNK, and P38. And immunohistochemistry staining was performed to evaluate the levels of estrogen receptor α (ERα), progesterone receptor (PR), nuclear factor-(NF-)κB (p65), interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), cyclooxygenases 2 (COX-2), inducible nitric oxide synthase (iNOS), 8-hydroxy-2'deoxyguanosine (8-OHdG), and nitrotyrosine (NT). Our results indicate that LRK treatment rescues significantly nipples height and diameter, decreases uterus index and ameliorates HMG. LRK treatment also markedly attenuates the over-expression of IL-1ß, TNF-α, COX-2, and iNOS, and suppressed the formation of 8-OHdG and NT. Furthermore, LRK treatment significantly inhibits the phosphorylation of JNK, ERK, and p38 and expression of NF-κB (p65), interestingly, LRK treatment has no effect on the expression of ERα and PR. Our data suggest that the LRK treatment protects the mammary glands from the damage of oxidative stress and inflammation induced by estrogen and progesterone, via suppresses of MAPK/NF-κB signaling pathways without affecting on the expression of ERα and PR.

Therapeutic Effects of a Traditional Chinese Medicine Formula Plus Tamoxifen vs. Tamoxifen for the Treatment of Mammary Gland Hyperplasia: A Meta-Analysis of Randomized Trials.

As a common disorder that accounts for over 70% of all breast disease cases, mammary gland hyperplasia (MGH) causes a severe problem for the quality of patients' life, and confers an increased risk of breast carcinoma. However, the etiology and pathogenesis of MGH remain unclear, and the safety and efficacy of current western drug therapy for MGH still need to be improved. Therefore, a meta-analysis was conducted by our team to determine whether a TCM formula named Ru-Pi-Xiao in combination with tamoxifen or Ru-Pi-Xiao treated alone can show more prominent therapeutic effects against MGH with fewer adverse reactions than that of tamoxifen. Studies published before June 2017 were searched based on standardized searching rules in several mainstream medical databases. A total of 27 articles with 4,368 patients were enrolled in this meta-analysis. The results showed that the combination of Ru-Pi-Xiao and tamoxifen could exhibit better therapeutic effects against MGH than that of tamoxifen (OR: 3.79; 95% CI: 3.09-4.65; P < 0.00001) with a lower incidence of adverse reactions (OR: 0.35; 95% CI: 0.28-0.43; P < 0.00001). The results also suggested that this combination could improve the level of progesterone (MD: 2.22; 95% CI: 1.72-2.71; P < 0.00001) and decrease the size of breast lump (MD: -0.67; 95% CI: -0.86 to -0.49; P < 0.00001) to a greater extent, which might provide a possible explanation for the pharmacodynamic mechanism of Ru-Pi-Xiao plus tamoxifen. In conclusion, Ru-Pi-Xiao and related preparations could be recommended as auxiliary therapy combined tamoxifen for the treatment of MGH.

Paeoniflorin ameliorates cholestasis via regulating hepatic transporters and suppressing inflammation in ANIT-fed rats.

Paeoniflorin has shown the obvious effect on cholestasis according to our previous research. However, its mechanism has not been absolutely explored yet. This study aims at evaluating the potential effect of paeoniflorin on alpha-naphthylisothiocyanate (ANIT) -induced cholestasis by inhibiting nuclear factor kappa-B (NF-κB) and simultaneously regulating hepatocyte transporters. Cholestasis was induced by administration of ANIT. The effect of paeoniflorin on serum indices such as total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltranspeptidase (γ-GT), total bile acid (TBA) and histopathology of liver were determined. Liver protein levels of NF-κB, interleukin 1ß (IL-1ß) and the hepatocyte transporters such as Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2) and cholesterol 7α-hydroxylase (Cyp7a1) were investigated by western blotting. The results demonstrated that paeoniflorin could decrease serum ALT, AST, ALP, γ-GT, TBIL, DBIL and TBA in ANIT-treated rats. Histological examination revealed that rats treated with paeoniflorin represented fewer neutrophils infiltration, edema and necrosis in liver tissue compared with ANIT rats. Moreover, paeoniflorin significantly reduced the over expressions of NF-κB and IL-1ß induced by ANIT in liver tissue. In addition, the relative protein expressions of NTCP, BSEP, MRP2 but not Cyp7a1 were also restored by paeoniflorin. The potential mechanism of paeoniflorin in alleviating ANIT-induced cholestasis seems to be related to reduce the over expressions of NF-κB and hepatocyte transporters such as NTCP, BSEP as well as MRP2.

[Acupuncture with regulating menstruation to promote pregnancy for diminished ovarian reverse: a prospective case series study].

OBJECTIVE: To investigate the effect and safety of acupuncture with regulating menstruation to promote pregnancy for diminished ovarian reverse (DOR). METHODS: According to prospective case series, 46 patients were observed and finally 40 cases were included. The acupoints were ① Baihui (GV 20), Shenting (GV 24), Guanyuan (CV 4) and bilateral Benshen (GB 13), Huangshu (KI 16), Dahe (KI 12), Luanchao (Extra), Zusanli (ST 36), Sanyinjiao (SP 6), Taixi (KI 3), Taichong (LR 3) and ② bilateral Shenshu (BL 23) and Ciliao (BL 32). The points in the two groups were used alternately. Acupuncture was given for 3 courses, 12 times as a course and 3 times a week. Before and after treatment, and 3 months after treatment, follicle-stimulating hormone (FSH), follicle-stimulating hormone/luteinizing hormone (FSH/LH), estradiol (E2), antral follicle count (AFC) and TCM symptom score were observed. The safety was evaluated. RESULTS: Compared with before treatment, the levels of FSH, FSH/LH decreased, and the levels of E2 and AFC increased after treatment and at follow-up (all P<0.05). And the TCM symptom scores were significantly lower than those before treatment (both P<0.05). The rate of pregnancy after treatment was 15% (6/40). There was no infection and organ injury. CONCLUSION: Acupuncture with regulating menstruation to promote pregnancy can safely improve the ovarian reserve of patients with DOR.

In Vivo Metabolite Profiling of a Purified Ellagitannin Isolated from Polygonum capitatum in Rats.

Ellagitannin is a common compound in food and herbs, but there are few detailed studies on the metabolism of purified ellagitannins. FR429 is a purified ellagitannin with antitumor potential, which is from Polygonum capitatum Buch.-Ham.ex D. Don. The present study was designed to investigate the metabolic profiles of FR429 in rats in vivo. Using liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MS(n)-IT-TOF), total eight metabolites were found in rat bile and urine after intravenous administration of FR429, but could not be detected in plasma. These metabolites were ellagic acid, mono-methylated FR429, ellagic acid methyl ether glucuronide, ellagic acid methyl ether diglucuronide, ellagic acid dimethyl ether glucuronide, and ellagic acid dimethyl ether diglucuronide. It was concluded that methylation and subsequent glucuronidation were the major metabolic pathways of FR429 in rats in vivo. This is the first report on the in vivo metabolism of the purified ellagitannin in rats.

In vivo and in vitro anti-inflammatory effects of Zao-Jiao-Ci (the spine of Gleditsia sinensis Lam.) aqueous extract and its mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Zao-Jiao-Ci (ZJC), as the spine of Chinese Honey locust (Gleditsia sinensis Lam.), is traditionally used as Chinese medicine to reduce inflammation. AIM OF THE STUDY: The present study aimed to investigate an anti-inflammatory effect of ZJC aqueous extract both in vitro and in vivo, as well as its underlying mechanisms. MATERIALS AND METHODS: Anti-inflammatory effect of ZJC aqueous extract was evaluated by using carrageenan-induced paw edema in rats. In addition, the inhibitory effects of ZJC on nitric oxide production, intracellular reactive oxygen species production, pro-inflammatory mediator expression and prostaglandin E2 (PGE2) production were determined by using LPS-activated RAW 264.7 cells. The anti-oxidant activity of ZJC was assessed using 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid assay. RESULTS: ZJC aqueous extract showed significant suppressive effect on paw edema in rats at 100mg/kg. Moreover, ZJC aqueous extract decreased the expression of cyclooxygenase (COX)-2 and significantly decreased the PGE2, tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 production in LPS-activated macrophages in dose-dependent manners. ZJC aqueous extract inhibited the mRNA expression of these inflammatory cytokines as well. Furthermore, ZJC aqueous extract was found as an anti-oxidant and could inhibit ROS production in the LPS-induced cells. CONCLUSIONS: These findings show the potential of ZJC aqueous extract as a naturally occurring COX-2 inhibitor to reduce inflammation.

Formononetin promotes angiogenesis through the estrogen receptor alpha-enhanced ROCK pathway.

Formononetin is an isoflavone that has been shown to display estrogenic properties and induce angiogenesis activities. However, the interrelationship between the estrogenic properties and angiogenesis activities of formononetin are not well defined. In the present study, docking and enzymatic assay demonstrated that formononetin displayed direct binding to the ligand-binding domain (LBD) of estrogen receptor alpha (ERα) with an agonistic property. Results from Human Umbilical Vein Endothelial Cells (HUVEC) by using real-time migration xCELLigence system, immunofluorescence and western blotting provided strong evidences of formononetin induced endothelial cell migration and dramatic actin cytoskeleton spatial modification through ERα-enhanced-ROCK-II/MMP2/9 signaling pathways. In addition, results from co-immunoprecipitation suggested formononetin induced cell migration via recruiting of ERα/ROCK-II activated complex formation. More interestingly, in zebrafish embryo we observed that formononetin significantly promoted angiogenic sproutings in the subintestinal vessels (SIVs) that could be completely abolished by ROCK inhibitor. In this study, we elucidated the underlying mechanisms that formononetin produced proangiogenesis effects through an ERα-enhanced ROCK-II signaling pathways. Results from the present study also expand our knowledge about the enigmatic underlying mechanisms of phytoestrogenic compounds in the promotion of angiogenesis in relation to ERα and ROCK interaction in endothelial cells and their relationship with actin assembly and cell migration.

Miltirone Is a Dual Inhibitor of P-Glycoprotein and Cell Growth in Doxorubicin-Resistant HepG2 Cells.

Miltirone (1), an abietane-type diterpene quinone isolated from Salvia miltiorrhiza, possesses anticancer activity in p-glycoprotein (P-gp)-overexpressing human cancer cells. Results of the current study suggest a dual effect of miltirone on P-gp inhibition and apoptotic induction in a human hepatoma HepG2 cell line and its P-gp-overexpressing doxorubicin-resistant counterpart (R-HepG2). Miltirone (1) elicited a concentration-dependent cytotoxicity, with a similar potency (EC50 ≈ 7-12 µM), in HepG2 and R-HepG2 cells. Miltirone (1) (1.56-6.25 µM) produced synergistic effects on doxorubicin (DOX)-induced growth inhibition of R-HepG2 (synergism: 0.3 < combination index < 0.5). Molecular docking studies illustrated that miltirone (1) interacted with the active site of P-gp with a higher binding affinity than DOX, suggesting that it was a P-gp inhibitor. Flow cytometric analysis confirmed miltirone (1) as a competitive inhibitor of P-gp. At non-necrotic concentrations (1.56-25 µM), miltirone (1) activated caspase-dependent apoptotic pathways and triggered the generation of reactive oxygen species (ROS) and ROS-mediated mitogen-activated protein kinase (MAPK) signaling pathways (e.g., p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase, and extracellular regulated kinase 1/2) in both HepG2 and R-HepG2 cells. Thus, we conclude that miltirone (1) is a dual inhibitor of P-gp and cell growth in human drug-resistant hepatoma cells.

Danshen (Salvia miltiorrhiza) water extract inhibits paracetamol-induced toxicity in primary rat hepatocytes via reducing CYP2E1 activity and oxidative stress.

OBJECTIVES: This study aimed to investigate the protective effects of Danshen (Salvia miltiorrhiza) water extract (DSE) and its major phenolic acid components against CYP2E1-mediated paracetamol (APAP)-induced hepatic toxicity. METHODS: The protection and underlying mechanisms were detected in CYP2E1 overexpression primary rat hepatocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alamar blue assay, CYP2E1 inhibition assay and glutathione assay. KEY FINDINGS: After APAP treatment, DSE (0.06-1 mg/ml) significantly increased cell viability in MTT assay. Two major components danshensu (8.2-130.5 µm) and salvianolic acid B (Sal B; 3.3-53.5 µm) mainly contributed to this protection, but rosmarinic acid, protocatechuic aldehyde and Sal A did not. Alamar blue assay showed that DSE, danshensu and Sal B maintained mitochondrial metabolic activity. DSE inhibited CYP2E1 (Ki = 1.46 mg/ml) in a mixed mode in rat liver microsomes in vitro; DSE decreased APAP-induced total glutathione depletion and preserved redox status (GSH/GSSG ratio) in hepatocytes. Danshensu and Sal B did not inhibit CYP2E1 or decrease total glutathione depletion, but preserved redox status. CONCLUSIONS: DSE protected hepatocytes against APAP-induced injury via maintenance of mitochondrial metabolic activity, CYP2E1 inhibition, reduction of total glutathione depletion and preservation of redox status. Danshensu and Sal B were mainly responsible for this protection.