Role of Fibrinogen-like Protein 1 in Tumor Recurrence Following Hepatectomy.

Partial hepatectomy is a first-line treatment for hepatocellular carcinoma. Within 2 weeks following partial hepatectomy, specific molecular pathways are activated to promote liver regeneration. Nevertheless, residual microtumors may also exploit these pathways to reappear and metastasize. Therapeutically targeting molecules that are differentially regulated between normal cells and malignancies, such as fibrinogen-like protein 1 (FGL1), appears to be an effective approach. The potential functions of FGL1 in both regenerative and malignant cells are discussed within the ambit of this review. While FGL1 is normally elevated in regenerative hepatocytes, it is normally downregulated in malignant cells. Hepatectomy does indeed upregulate FGL1 by increasing the release of transcription factors that promote FGL1, including HNF-1α and STAT3, and inflammatory effectors, such as TGF-ß and IL6. This, in turn, stimulates certain proliferative pathways, including EGFR/Src/ERK. Hepatectomy alters the phase transition of highly differentiated hepatocytes from G0 to G1, thereby transforming susceptible cells into cancerous ones. Activation of the PI3K/Akt/mTOR pathway by FGL1 allele loss on chromosome 8, a tumor suppressor area, may also cause hepatocellular carcinoma. Interestingly, FGL1 is specifically expressed in the liver via HNF-1α histone acetylase activity, which triggers lipid metabolic reprogramming in malignancies. FGL1 might also be involved in other carcinogenesis processes such as hypoxia, epithelial-mesenchymal transition, immunosuppression, and sorafenib-mediated drug resistance. This study highlights a research gap in these disciplines and the necessity for additional research on FGL1 function in the described processes.

Monitoring norepinephrine release in vivo using next-generation GRABNE sensors.

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter. The first-generation NE sensor makes in vivo, real-time, cell-type-specific and region-specific NE detection possible, but its low NE sensitivity limits its utility. Here, we developed the second-generation GPCR-activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response and high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell-type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

Improved green and red GRAB sensors for monitoring dopaminergic activity in vivo.

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a large network of dopaminergic projections. To dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent G-protein-coupled receptor activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity and signal-to-noise ratio with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

Beneficial effects of silibinin on serum lipids, bile acids, and gut microbiota in methionine-choline-deficient diet-induced mice.

Background and purpose: Silibinin (SIL) is a flavonoid lignin isolated from the fruit and seeds of silybum marianum that exhibits good therapeutic potential for NASH. However, the effects of SIL on serum lipids, bile acids (BAs), and gut microbiota (GM) in NASH mice remain unknown. The present work aimed to explore the beneficial effects of SIL supplementation on serum lipids, bile acids, and gut microbiota in MCD mice. Experimental approach: After male C57BL/6 mice were fed with a methionine-choline deficient (MCD) diet and simultaneously gavaged with SIL (20 mg/kg. d) for 8 weeks, the pathological changes of liver tissue were observed by oil red O, haematoxylin-eosin, and Masson tricolor staining; the levels of serum AST and ALT, and liver TG and MDA were detected by assay kits; metabonomics and 16S rDNA sequencing were used to analyze the composition of serum lipids and BAs and the abundance of GM; and the mRNA expression levels of hepatic genes related to BAs homeostasis were detected by RT-qPCR. Results: The results indicated that SIL treatment decreased the levels of 26 lipids (including four arachidonic acids, seven FFAs, 12 acyl carnitines, and three GPs) and two BAs (23-DCA, GLCA), while Dubosiella increased the levels of 10 lipids (including TxB3, PG16:0_18:1, Cer t18:0/24:0 and 7 TGs), five BAs (ß-MCA, α-MCA, UDCA, 3-oxo-DCA and HCA), and two GMs (Verrucomicrobiota and Akkermansiaceae) of MCD mice, but had no significant effect on the mRNA expression of CYP7A1, CYP27A1, Bsep, Mrp2, Ntcp, or Oatp1b2. Therefore, influencing GM composition and then regulating the levels of serum lipids and BAs through enterohepatic axis should be an important mechanism of SIL-induced alleviative effect on MCD mice. More importantly, we found that SIL had a good coordination in regulating the abundance of GM and the contents of serum lipids and BAs in MCD mice, that is, when the abundance of probiotics was up-regulated, the content of beneficial unsaturated fatty acids in serum was up-regulated, while the serum levels of harmful lipids and BAs were down-regulated. Conclusion: The alleviating effect of SIL on NASH may be closely related to the correction of intestinal bacteria disorder, serum bile acid, and lipid metabolic disturbance in mice.

Improved dual-color GRAB sensors for monitoring dopaminergic activity in vivo.

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a vast network of dopaminergic projections. To fully dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent GPCR activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity, and signal-to-noise properties with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in freely moving mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala, and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

Dihydromyricetin ameliorated nonalcoholic steatohepatitis in mice by regulating the composition of serous lipids, bile acids and ileal microflora.

BACKGROUND: Dihydromyricetin (DMY) is a natural flavonoid with anti-nonalcoholic steatohepatitis (NASH) activity. However, the effects of DMY on the composition of lipids and bile acids (BAs) in serum, and gut microbiota (GM) in ileum of mice with NASH are not clear. METHODS: After male C57BL/6 mice was fed with methionine and choline deficiency (MCD) diet and simultaneously administered with DMY (300 mg/kg/day) by gavage for 8 weeks, the pathological changes of liver tissue were observed by Oil Red O, hematoxylin eosin and Masson staining, the levels of serum alaninea minotransferase, aspartate aminotransferase and liver triglyceride, malonic dialdehyde were detected by the detection kits, the composition and contents of serum lipids and BAs were detected by Liquid Chromatograph-Mass Spectrometry, the mRNA levels of hepatic BAs homeostasis-related genes were detected by RT-qPCR, and microbiological diversity in ileum was analyzed by 16S rDNA sequencing. RESULTS: The results showed that the significant changes including 29 lipids, 4 BAs (23-nor-deoxycholic acid, ursodeoxycholic acid, 7-ketodeoxycholic acid and cholic acid), 2 BA transporters (Mrp2 and Oatp1b2) and 8 GMs between MCD and DMY groups. Among them, DMY treatment significantly down-regulated 21 lipids, 4 BAs mentioned above, the ratio of Firmicutes/Bacteroidota and the abundance of Erysipelotrichaceae, Faecalibacuium, significantly up-regulated 8 lipids and 5 GMs (Verrucomicrobiota, Bacteroidota, Actinobacteria, Akkermansiaceae and Akkermansia). CONCLUSIONS: The results suggested that DMY may alleviate MCD diet-induced NASH through decreasing the serum levels of toxic BAs which regulated by liver Oatp1b2 and Mrp2, regulating the metabolism of related lipids, and up-regulating intestinal probiotics (Actinobacteria and Verrucomicrobiota at the phylum level; Akkermansiaceae at the family level; Akkermansiaat at the genus level) and inhibiting intestinal harmful bacteria (Firmicutes at the phylum level; Erysipelotrichaceae at the family level; Faecalibaculum at the genus level).

Intrinsic dopamine and acetylcholine dynamics in the striatum of mice.

External rewards such as food and money are potent modifiers of behaviour1,2. Pioneering studies established that these salient sensory stimuli briefly interrupt the tonic discharge of neurons that produce the neuromodulators dopamine (DA) and acetylcholine (ACh): midbrain DA neurons (DANs) fire a burst of action potentials that broadly elevates DA in the striatum3,4 at the same time that striatal cholinergic interneurons (CINs) produce a characteristic pause in firing5,6. These phasic responses are thought to create unique, temporally limited conditions that motivate action and promote learning7-11. However, the dynamics of DA and ACh outside explicitly rewarded situations remain poorly understood. Here we show that extracellular DA and ACh levels fluctuate spontaneously and periodically at a frequency of approximately 2 Hz in the dorsal striatum of mice and maintain the same temporal relationship relative to one another as that evoked by reward. We show that this neuromodulatory coordination does not arise from direct interactions between DA and ACh within the striatum. Instead, we provide evidence that periodic fluctuations in striatal DA are inherited from midbrain DANs, while striatal ACh transients are driven by glutamatergic inputs, which act to locally synchronize the spiking of CINs. Together, our findings show that striatal neuromodulatory dynamics are autonomously organized by distributed extra-striatal afferents. The dominance of intrinsic rhythms in DA and ACh offers new insights for explaining how reward-associated neural dynamics emerge and how the brain motivates action and promotes learning from within.

Honokiol attenuates acetaminophen-induced acute liver injury by inhibiting hepatic CYP1A2 activity and improving liver mitochondrial dysfunction.

Objective: Acetaminophen (APAP) overdose is a common cause of liver injury. This study aimed to investigate the protective effect of honokiol (Hon) against APAP-induced hepatotoxicity and its potential mechanism. Methods: C57BL/6 mice were administrated with Hon (10 and 30 mg/kg) after APAP (300 mg/kg) treatment. On 1.5 h and 5 h after Hon treatment, mice were sacrificed. Serum and liver were collected. And then, liver injury-related indexes, APAP metabolism-related indexes, mitochondrial respiratory chain function-related indexes, and mitochondrial membrane function-related protein expression were evaluated. Results: It was found that Hon significantly decreased serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) activity and glutathione (GSH) depletion, increased hepatic catalase (CAT) and GSH peroxidase (GSH-Px) activities, reduced hepatic MDA and 3-nitrotyrosine contents, inhibited hepatic CYP1A2 activity and APAP protein adducts (APAP-CYS) formation. Meanwhile, oxidative phosphorylation capacity of complex I and electron transfer capacity of complex IV in mitochondrial respiratory chain was increased, whereas the release of H2O2 in the mitochondria was decreased following Hon treatment. Furthermore, Hon markedly down-regulated p-JNK in both cytosol and mitochondria, and obviously inhibited the release of apoptosis inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to cytosol. Conclusion: Hon alleviated APAP-induced liver injury through the following pathways: Reducing the production of APAP-CYS by inhibiting CYP1A2 activity; Ameliorating hepatic oxidative stress by increasing the levels of hepatic CAT, GSH-Px and GSH; Improving mitochondrial respiratory chain function by promoting oxidative phosphorylation capacity of complex I and electron transfer capacity of complex IV; Improving the function of mitochondrial membrane by inhibiting p-JNK and its translocation to mitochondria, thereby reducing the release of AIF and EndoG.

Dopaminergic System in Promoting Recovery from General Anesthesia.

Dopamine is an important neurotransmitter that plays a biological role by binding to dopamine receptors. The dopaminergic system regulates neural activities, such as reward and punishment, memory, motor control, emotion, and sleep-wake. Numerous studies have confirmed that the dopaminergic system has the function of maintaining wakefulness in the body. In recent years, there has been increasing evidence that the sleep-wake cycle in the brain has similar neurobrain network mechanisms to those associated with the loss and recovery of consciousness induced by general anesthesia. With the continuous development and innovation of neurobiological techniques, the dopaminergic system has now been proved to be involved in the emergence from general anesthesia through the modulation of neuronal activity. This article is an overview of the dopaminergic system and the research progress into its role in wakefulness and general anesthesia recovery. It provides a theoretical basis for interpreting the mechanisms regulating consciousness during general anesthesia.

Synthesis of 4-Tetrazolyl-Substituted 3,4-Dihydroquinazoline Derivatives with Anticancer Activity via a One-Pot Sequential Ugi-Azide/Palladium-Catalyzed Azide-Isocyanide Cross-Coupling/Cyclization Reaction.

A new one-pot preparation of 4-tetrazolyl-3,4-dihydroquinazolines has been reported. The Ugi-azide reactions of 2-azidobenzaldehydes, amines, trimethylsilyl azide, and isocyanides produced azide intermediates without separation, which were treated with isocyanides to give 4-tetrazolyl-3,4-dihydroquinazoline derivatives through a sequential Palladium-catalyzed azide-isocyanide cross-coupling/cyclization reaction in moderate to good yields. The biological evaluation demonstrated that compound 6c inhibited breast cancer cells well and displayed broad applications for synthesis and medicinal chemistry.

The determination of monosaccharide in different years Qingzhuan Dark Tea polysaccharide by liquid chromatography-mass spectrometry.

AIM: To establish a fast, sensitive and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for determining the monosaccharide content of Qingzhuan Dark Tea polysaccharides in different years (2 years, 5 years and 11 years). METHODS: The optimised chromatographic conditions were achieved on a C18 column (5.0 µm, 250 mm × 4.6 mm inner diameter). The mobile phase flow rate was 0.9 mL/min and the column temperature was set to 27°C. The aqueous phase A (5 mM aqueous ammonium acetate) and organic phase B (acetonitrile) were used to elute the target analyses isocratically (0-60 min: 18% B). The mass spectrometer detector was equipped with an electron spray ionisation (ESI)source, and multiple reaction monitoring (MRM) mode was used for the determination of 1-phenyl-3-methyl-5-pyrazolone (PMP) derived monosaccharides. RESULTS: We carried out a comprehensive methodological validation of PMP derived monosaccharides, including linearity, precision, stability and repeatability. Nine monosaccharides (rhamnose, mannose, ribose, glucose, galacturonic acid, xylose, galactose, fucose and arabinose) of Qingzhuan Dark Tea polysaccharides were identified, in which ribose and fucose were reported for the first time. The results showed the contents of these nine monosaccharides differed significantly among different years. CONCLUSIONS: The validated method is reliable, accurate, repeatable and can be applied to quality assessment of these monosaccharides.

Intravenous versus inhalational maintenance of anesthesia for quality of recovery in adult patients undergoing non-cardiac surgery: A systematic review with meta-analysis and trial sequential analysis.

BACKGROUND: Intravenous and inhalational agents are commonly used in general anesthesia. However, it is still controversial which technique is superior for the quality of postoperative recovery. This meta-analysis aimed at comparing impact of total intravenous anesthesia (TIVA) versus inhalational maintenance of anesthesia on the quality of recovery in patients undergoing non-cardiac surgery. METHODS: We systematically searched EMBASE, PubMed, and Cochrane library for randomized controlled trials (RCTs), with no language or publication status restriction. Two authors independently performed data extraction and assessed risk of bias. The outcomes were expressed as mean difference (MD) with 95% confidence interval (CI) based on a random-effect model. We performed trial sequential analysis (TSA) for total QoR-40 scores and calculated the required information size (RIS) to correct the increased type I error. RESULTS: A total of 156 records were identified, and 9 RCTs consisting of 922 patients were reviewed and included in the meta-analysis. It revealed a significant increase in total QoR-40 score on the day of surgery with TIVA (MD, 5.91 points; 95% CI, 2.14 to 9.68 points; P = 0.002; I2 = 0.0%). The main improvement was in four dimensions, including "physical comfort", "emotional status", "psychological support" and "physical independence". There was no significant difference between groups in total QoR-40 score (P = 0.120) or scores of each dimension on POD1. The TSA showed that the estimated required information size for total QoR-40 scores was not surpassed by recovered evidence in our meta-analysis. And the adjusted Z-curves did not cross the conventional boundary and the TSA monitoring boundary. CONCLUSION: Low-certainty evidence suggests that propofol-based TIVA may improve the QoR-40 score on the day of surgery. But more evidence is needed for a firm conclusion and clinical significance.

In vitro metabolism of 4, 5-dimethoxycanthin-6-one by human liver microsomes and its inhibition on human CYP1A2.

AIMS: P. quassioides is a traditional Chinese medicine used for the treatment of gastroenteritis, snakebite, infection and hypertension in China. 4, 5-dimethoxycanthin-6-one is one of the main active canthinone alkaloid isolated from P. quassioides. The aim of this work was to identify the cytochrome P (CYP) 450 enzymes responsible for the metabolism of 4, 5-dimethoxycanthin-6-one (DCO) and to evaluate the inhibitory effect of DCO on CYP activity in human liver microsomes (HLM) in vitro. MATERIALS AND METHODS: the CYP isoforms responsible for DCO metabolism and the inhibitory effects of DCO on CYP activity was studied in HLM. KEY FINDINGS: The in vitro metabolic enzyme of DCO was CYP3A4 (mediated the formation of metabolites M1-M5), CYP2C9 (mediated the formation of metabolites M1-M3, M6 and M8) and CYP2D6 (mediated the formation of metabolite M3) in HLM. Furthermore, the present work found that DCO uncompetitively inhibited CYP1A2-mediated phenacetin O-deethylation with an IC50 value of 1.7µM and a Ki value of 2.6µM. SIGNIFICANCE: The results suggested that the metabolic interaction should be existed when the substrate drugs of CYP1A2 were co-administered with DCO or traditional Chinese medicine containing it, such as the extract of P. quassioides and Kumu injection.

Pharmacokinetics and tissue distribution of 4,5-dimethoxycanthin-6-one and its major metabolites in rats.

4,5-Dimethoxycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the active ingredients of P. quassiodes. In the present work, a LC-MS/MS method was developed for the determination of 4,5-dimethoxycanthin-6-one and its major metabolites 5-hydroxy-4-methoxycanthin-6-one (M1) and 4-hydroxy-5-methoxycanthin-6-one (M2) in rat plasma and tissues, and applied to study their pharmacokinetics and tissue distribution after intramuscular administration of 4,5-dimethoxycanthin-6-one to rats. By protein precipitation with methanol for plasma samples and liquid-liquid extraction with ethyl acetate for tissue samples, the analytes were separated on an ODS C18 column with a mobile phase consisted of methanol and water (0.1% formic acid), and quantified by a MS detector in positive multiple reaction monitoring (MRM) mode. MS transitions were m/z 281.0→167.1 for 4,5-dimethoxycanthin-6-one, m/z 267.0→168.1 for M1 and M2, m/z 251.0→195.1 for 3-methylcanthin-2,6-dione (IS). The pharmacokinetic results indicate that 4,5-dimethoxycanthin-6-one is absorbed rapidly (Tmax=5.4-6.4min), distributed rapidly and widely in the order of liver>kidney≈lung≈large intestine≈small intestine, and eliminated quickly (t1/2z=64.9-77.7min) following the intramuscular administration. Furthermore, M1 and M2 were detected only in rat plasma and liver at the indicated times after the intramuscular administration.

[Degradation kinetics of chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid at neutral and alkaline pH values].

The degradation kinetics of chlorogenic acid (5-CQA), cryptochlorogenic acid (4-CQA), and neochlorogenic acid (3-CQA) in aqueous solution at 37 degrees C and different pH values (7.05, 7.96, 9.25) were investigated in the present work. The results indicated that 3-, 4- and 5-CQA tended to remain stable in acidic pH circumstance, and unstable in neutral and alkaline pH circumstance. With the increase of the alkalinity, the degradation of 3-, 4- and 5-CQA was increased leading to a less amount of total CQA and was satisfactorily described by the Weibull equation. Meanwhile, caffeic acid was not detected after the degradation of CQA. Moreover, the degradation of 3-CQA and 5-CQA tended to be converted to 4-CQA, and the degradation of 4-CQA tended to be converted to 3-CQA rather than 5-CQA. The comparison of the degradation kinetics parameters of 3-, 4- and 5-CQA at neutral and alkaline pH values showed that the orders of the rate constant (k) values were 4-CQA > 3-CQA > 5-CQA, while the orders of the degradation half life (t½) values were 4-CQA < 3-CQA < 5-CQA, indicating the orders of the stabilities of 3-, 4- and 5-CQA at 37 degrees C and neutral and alkaline pH values were 4-CQA < 3-CQA < 5-CQA.

Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-one by HPLC-Q-TOF-MS/MS.

4,5-Dimthexycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the main active ingredients of Picrasma quassioides, which is a widely used herbal medicine for the treatment of gastroenteritis, snakebite, infection and hypertension in China. In the present study, the in vitro metabolites of 4,5-dimethoxycanthin-6-one in rat, mouse, dog and human liver microsomes, as well as the in vivo metabolites in rat plasma and urine following a single oral dose of 4,5-dimethoxycanthin-6-one, were identified by high-performance liquid chromatography combined with triple TOF mass spectrometry (HPLC-TOF/MS/MS). The metabolites were elucidated based on an accurate mass measurement, the MS/MS fragmentation patterns, the retention times of the parent drug and its metabolites, and the relevant drug biotransformation rules. After incubation in liver mcrosomes for 50 min, 4,5-dimethoxycanthin-6-one produced 8 phase I metabolites including 2 mono-demethylated metabolites (M1, M2), 3 mono-hydroxylated metabolites (M3-M5), and 3 mono-demethylated and mono-hydroxylated metabolites (M6-M8) in rat and mouse liver microsomes, 7 phase I metabolites (without M7) in dog and human liver microsomes. After a single oral administration of 4,5-dimethoxycanthin-6-one to rats, there were 3 phase I metabolites (M1, M2 and M5) detected in rat plasma and 5 phase I metabolites (M1-M5) in rat urine. Phase II metabolites were not detected in rat plasma and urine. Among these metabolites, mono-demethylated metabolites (M1 and M2) were the major metabolites of 4,5-dimethoxycanthin-6-one, mono-hydroxylated metabolites (M3-M5) were the minor metabolites of 4,5-dimethoxycanthin-6-one.

The pharmacokinetics and bioavailability of three canthinone alkaloids after administration of Kumu injection to rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kumu injection (KMI) is made from the branches and stems of Picrasma quassiodes (D. Don) Benn. and has been used clinically for the treatment of upper respiratory tract infection, acute tonsillitis, enteritis and bacillary dysentery. 3-methylcanthin-2,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 4,5-dimethoxycanthin-6-one are the active ingredients of KMI because of its therapeutic effects. AIM OF THE STUDY: To develop a LC-MS/MS method for simultaneous determination of three active canthinone alkaloids (4,5-dimethoxycanthin-6-one, 5-hydroxy-4-methoxycanthin-6-one and 3-methylcanthin-2,6-dione) in rat plasma and for the pharmacokinetic study of them after administered of KMI to rats. MATERIALS AND METHODS: Rats were divided into 5 groups (n=5 per group), 3 groups administered intramuscularly with a single dose of KMI at 0.30, 0.45 and 0.90mL/kg respectively, and the other 2 groups administered intragastically or intravenously a single dose of KMI at 0.9mL/kg respectively. The concentrations of 4,5-dimethoxycanthin-6-one, 5-hydroxy-4-methoxycanthin-6-one and 3-methylcanthin-2,6-dione in plasma were determined by the established LC-MS/MS method at different time points and the pharmacokinetic parameters were estimated by non-compartmental analysis. RESULTS: Pharmacokinetic results indicated that all of the alkaloids were absorbed rapidly and 3-methylcanthin-2,6-dione was eliminated fastest in rats. After intramuscular administration of KMI to rats, the absolute bioavailability is excellent, and the pharmacokinetic profiles are characterized by the first order kinetics. CONCLUSION: The established method is suitable for the quantitation of the three alkaloids in rat plasma. And this pharmacokinetic study suggested that intramuscular injection of KMI was suitable in clinical usage.

Mild Hypothermia Combined with Hydrogen Sulfide Treatment During Resuscitation Reduces Hippocampal Neuron Apoptosis Via NR2A, NR2B, and PI3K-Akt Signaling in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

We investigated whether mild hypothermia combined with sodium hydrosulfide treatment during resuscitation improves neuron survival following cerebral ischemia-reperfusion injury beyond that observed for the individual treatments. Male Sprague-Dawley rats were divided into seven groups (n = 20 for each group). All rats underwent Pulsinelli 4-vessel occlusion. Ischemia was induced for 15 min using ligatures around the common carotid arteries, except for the sham group. Immediately after initiating reperfusion, the mild hypothermia (MH), sodium hydrosulfide (NaHS), hydroxylamine (HA), MH + NaHS, MH + HA, and ischemia-reperfusion (I/R) control groups received an intraperitoneal injection of saline, sodium hydrosulfide, hydroxylamine, sodium hydrosulfide, hydroxylamine, and saline, respectively, and mild hypothermia (32 to 33 °C) was induced in the MH, MH + NaHS, and MH + HA groups for 6 h. The levels of NR2A, NR2B, p-Akt, and p-Gsk-3ß in the hippocampus of the MH, NaHS, and MH + NaHS groups were higher than those in the I/R control group, with the highest levels observed in the MH + NaHS group (P < 0.05). Treatment with hydroxylamine reduced the levels of these proteins in the HA and MH + HA groups, compared with the I/R control and MH groups, respectively. The apoptotic index of the CA1 region of the hippocampus was 45.2, 66.5, 63.5, and 84.8 % in the MH + NaHS, MH, NaHS, and I/R control groups, respectively (P < 0.05), indicating that the combination treatment shifted the NR2A/NR2B balance in favor of synaptic neuron stimulation and phosphatidylinositol 3'-kinase (PI3K)/Akt signaling. The combination of mild hypothermia and sodium hydrosulfide treatment for resuscitation following ischemia-reperfusion injury was more beneficial for reducing hippocampal apoptosis and pathology than that of mild hypothermia or hydrogen sulfide treatment alone.

Hydrogen sulphide and mild hypothermia activate the CREB signaling pathway and prevent ischemia-reperfusion injury.

BACKGROUND: Both hydrogen sulphide (H2S) and mild hypothermia have been reported to prevent brain damage caused by reperfusion assault through regulating the N-methyl-D-aspartate receptor (NMDAR). However, the relationship between the two treatments and how they exert neuro-protective effects through NMDARs remain to be elucidated. METHODS: Transient cerebral ischemia was induced using the Pulsinelli four-vessel occlusion method. We used sodium hydrosulphide (NaHS) as the H2S donor. We randomly divided 100 Sprague-Dawley rats into five groups of 20: Sham operation group (Sh), normothermic (36-37 °C) ischemia group (NT), mild hypothermic (32-33 °C) ischemia group (mHT), normothermic ischemia combined with NaHS treatment group (NT + NaHS), and mild hypothermic ischemia combined with NaHS treatment group (mHT + NaHS). After 6 hrs of reperfusion, rats were decapitated and hippocampus samples were immediately collected. We measured NR2A (GluN1), NR2B (GluN2) and p-CREB protein levels using western blotting. We further analyzed BDNF mRNA expression by real-time PCR. Hematoxylin and eosin (HE) staining was used to examine pyramidal cell histology at the CA1 region. All statistical analyses were carried out by ANOVA and LSD t-test as implemented by the SPSS 13.0 software. RESULTS: In the four test groups with ischemia-reperfusion, hippocampal H2S concentration increased following treatment, and administration of NaHS further increased H2S levels. Moreover, administration of both NaHS and mild hypothermia resulted in up-regulation of NR2A and NR2B protein expressions, as well as p-CREB protein and BDNF mRNA levels. At the cellular level, NaHS and mild hypothermia groups exhibited lower damage caused by ischemia-reperfusion in the CA1 region of the hippocampus. The strongest protective effect was observed in rats treated with combined NaHS and mild hypothermia, suggesting their effects were additive. CONCLUSION: Our results support previous findings that hydrogen sulphide and mild hypothermia can prevent ischemia-reperfusion injury. Both treatments caused an up-regulation of NMDA receptors, as well as an elevation in p-CREB protein and BDNF mRNA levels. Thus, hydrogen sulphide and mild hypothermia may provide neuro-protective effect through activating the pro-survival CREB signaling pathway.