Betaine alleviates cognitive impairment induced by homocysteine through attenuating NLRP3-mediated microglial pyroptosis in an m6A-YTHDF2-dependent manner.

Dementia, with homocysteine (Hcy) as an important risk factor, is a severe public health problem in the aging society. Betaine serves as a methyl donor and plays an important role in reducing Hcy. However, the effects and mechanisms of betaine on Hcy-induced cognitive impairment remain unclear. Firstly, SD rats were injected with Hcy (400 µg/kg) through vena caudalis, and betaine (2.5 % w/v) was supplemented via drinking water for 14 days. Betaine supplementation could attenuate Hcy-induced cognitive impairment in the Y maze and novel object recognition tests by repairing brain injury. Meanwhile, microglial activation was observed to be inhibited by betaine supplementation using immunofluorescence and sholl analysis. Secondly, HMC3 cells were treated with betaine, which was found to decrease the ROS level, ameliorate cell membrane rupture, reduce the release of LDH, IL-18 and IL-1ß, and attenuate the damage of microglia to neurons. Mechanistically, betaine alleviates cognitive impairment by inhibiting microglial pyroptosis via reducing the expressions of NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-18 and IL-1ß. Betaine treatment can increase SAM/SAH ratio, confirming its enhancement on methylation capacity. Furthermore, betaine treatment was found to enhance N6-methyladenosine (m6A) modification of NLRP3 mRNA, and reduced the NLRP3 mRNA stability through increasing the expression of the m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Finally, silencing YTHDF2 could reverse the inhibitory effect of betaine on pyroptosis. Our data demonstrated that betaine attenuated Hcy-induced cognitive impairment by suppressing microglia pyroptosis via inhibiting the NLRP3/caspase-1/GSDMD pathway in an m6A-YTHDF2-dependent manner.

Betaine attenuates age-related suppression in autophagy via Mettl21c/p97/VCP axis to delay muscle loss.

Age-related impairment of autophagy accelerates muscle loss and lead to sarcopenia. Betaine can delay muscle loss as a dietary methyl donor via increasing S-adenosyl-L-methionine (SAM, a crucial metabolite for autophagy regulation) in methionion cycle. However, whether betaine can regulate autophagy level to attenuate degeneration in aging muscle remains unclear. Herein, male C57BL/6J young mice (YOU, 2-month-old), old mice (OLD, 15-month-old), and 2%-betaine-treated old mice (BET, 15-month-old) were employed and raised for 12 weeks. All mice underwent body composition examination and grip strength test before being sacrificed. Betaine alleviated age-related decline in muscle mass and strength. Meanwhile, betaine preserved the expression autophagy markers (Atg5, Atg7, LC3-II, and Beclin1) both at transcriptional and translational level during the aging process. RNA-sequencing results generated from mice gastrocnemius muscle found Mettl21c, a SAM-dependent autophagy-regulating methyltransferase, was significantly higher expressed in BET and YOU group. Results were further validated by qPCR and western bloting. In vitro, C2C12 cells with or without Mettl21c RNA interference were treated different concentration of betaine (0 mM, 10 mM) under methionine-starved condition. Compared with control group, betaine upregulated autophagy markers expression and autophagy flux. By increasing the SAM level, betaine facilitated trimethylation of p97 (Mettl21c downstream effector) into valosin-containing protein (VCP). Increased VCP promoted autophagic turnover of cellular components, ATP production, and cell differentiation. Knock-down of Metthl21c dismissed improvements mentioned above. Collectively, betaine could enhance aged skeletal muscle autophagy level via Mettl21c/p97/VCP axis to delay muscle loss.

Hezi inhibits Tiebangchui-induced cardiotoxicity and preserves its anti-rheumatoid arthritis effects by regulating the pharmacokinetics of aconitine and deoxyaconitine.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiebangchui (TBC, dried roots of Aconitum pendulum Busch. and Aconitum flavum Hand.-Mazz.) is a well-known Tibetan medicine for dispelling cold and relieving pain. In China, it is widely used in prevention and treatment of various diseases, such as rheumatoid arthritis (RA), traumatic injury, and fracture. However, its cardiotoxicity and neurotoxicity seriously restrict its clinical application. Traditionally, Hezi (HZ, dry ripe fruit of Terminalia chebula Retz. and Terminalia chebula Retz. var. tomentella Kurt.) is generally used in combination with TBC for the purpose of toxicity reducing and efficacy enhancing, but so far we still can't clearly elucidate the compatibility effect and mechanism of the classical herbal pair. AIM OF STUDY: To investigate the compatibility effect and mechanism of TBC co-administered with HZ. METHODS: In the present study, we clarified the cardioprotective role of HZ on the cardiotoxicity induced by TBC. The electrocardiogram, the levels of serum cardiac troponin T (cTnT), the activities of cardiac superoxide dismutase (SOD), malonaldehyde (MDA), and histopathology of heart tissue have been determined in each group. Meanwhile, the anti-RA effect of each group was investigated by paw swelling measurement and histopathological examination of synovial. To explore the underlying mechanism, we performed the pharmacokinetic studies of aconitine (AC) and deoxyaconitine (DE) in TBC group and TBC + HZ group by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) system. RESULTS: TBC co-administered with HZ could significantly inhibit the increased heart rate and the prolonged QTc interval induced by TBC (p < 0.01). And TBC + HZ group had lower levels of serum cTnT, cardiac MDA, and higher levels of cardiac SOD compared with TBC group (p < 0.01). In addition, the combination of TBC and HZ could preserve the anti-RA effect of TBC. Both TBC administration alone and TBC + HZ combination administration could effectively alleviate the paw swelling (p < 0.01). Furthermore, TBC co-administered with HZ could significantly decrease the area under the concentration-time curve (AUC(0-∞)) and maximum concentration (Cmax) of AC and DE comapred with TBC administration alone (p < 0.01 or p < 0.05). Meanwhile, it was observed that the time to reach the peak concentration (Tmax), elimination half-life (t1/2), mean retention time (MRT) of AC and DE in TBC group were significantly higher than those in TBC + HZ group (p < 0.01 or p < 0.05). CONCLUSIONS: TBC co-administered with HZ could reduce TBC-induced cardiotoxicty and preserve its anti-RA efficacy. The underlying mechanism is associated with the change of pharmacokinetic process of AC and DE.

Sennoside A is a novel inhibitor targeting caspase-1.

The assembly of inflammasomes drives caspase-1 activation, which further promotes proinflammatory cytokine secretion and downstream pyroptosis. The discovery of novel caspase-1 inhibitors is pivotal to developing new therapeutic means for inflammasome-involved diseases. In our present study, sennoside A (Sen A), a popular ingredient in multiple weight-loss medicines and dietary supplements, is found to potently inhibit the enzymatic activity of caspase-1 in vitro. Sen A considerably decreased IL-1ß production in macrophages stimulated by LPS plus ATP, nigericin or MSU as well as poly(dA:dT) transfection, and remedied ROS-involved pyroptosis via caspase-1 inhibition. Mechanistically, Sen A not only suppressed the assembly of both NLRP3 and AIM2 inflammasome but also affected the priming process of NLRP3 inflammasome by blocking NF-κB signaling. Sen A significantly ameliorated the pathophysiological effect in LPS-, MSU- and carrageenan-challenged rodent models by suppressing inflammasome activation. Furthermore, P2X7 was indispensable for Sen A inhibiting NLRP3 inflammasome since it failed to further decrease IL-1ß and IL-18 production in LPS plus ATP-stimulated BMDMs that were transfected with P2X7 siRNA. Sen A also restrained the large pore-forming functionalities of the P2X7R as verified by the YO-PRO-1 uptake assay. Taken together, Sen A inactivates caspase-1 to inhibit NLRP3 and AIM2 inflammasome-involved inflammation in a P2X7-dependent manner, making it an attractive candidate as a caspase-1 small-molecular inhibitor.

Effects of Citri Reticulatae Pericarpium and grapefruit juice on the pharmacokinetics of omeprazole in rats.

The effects of Citri Reticulatae Pericarpium (CRP) and grapefruit juice (GFJ) on the pharmacokinetics of omeprazole were investigated in this study. Sprague-Dawley rats were pretreated with CRP decoction or GFJ for 28 consecutive days. After a single intragastric administration of 6.0 mg/kg, the concentration of omeprazole in the plasma was determined by high-performance liquid chromatography (HPLC), and the pharmacokinetic parameters were calculated by Kinetica software 5.0. A high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) method was established to identify the chemical components in CRP decoction and GFJ. The results showed that the AUCt-∞ was significantly increased when coadministrated with CRP. The AUC0-t and AUC0-∞ was remarkably increased; the Cl was decreased when coadministrated with GFJ. A total of 31 and 28 bioactive compounds were identified in the CRP decoction and GFJ, respectively. Flavonoids and furanocoumarins, including hesperidin, hesperetin, naringenin, sinensetin, tangeretin, nobiletin, and 6',7'-dihydroxybergamottin, were simultaneously identified in CRP decoction and GFJ. This study indicates that the increased bioavailability of omeprazole may be due to the inhibition of hepatic cytochrome P450 enzymes, and the systemic exposure should be monitored when concomitant administration with CRP and GFJ. PRACTICAL APPLICATIONS: Citri Reticulatae Pericarpium (CRP) has been widely consumed as a daily condiment, functional food, and a traditional Chinese medicine. Omeprazole, primary metabolized by CYP450 enzymes, was usually coadministered with CRP for the treatment of gastrointestinal disease. Studies have confirmed that much fruit juices, including grapefruit juice, may affect drug metabolism enzymes. CRP and grapefruit (Citrus paradisi Macf.) belong to the genus Citrus and family Rutaceae with different species. Therefore, the pharmacokinetic interaction of CRP decoction and grapefruit juice with omeprazole is worthy of attention. The results of this study can provide basic pharmacological data support for the safe and effective clinical use of omeprazole. It can also provide a theoretical basis for the development of new functional products and daily application of CRP.

Benzaldehyde, A New Absorption Promoter, Accelerating Absorption on Low Bioavailability Drugs Through Membrane Permeability.

Styrax, one of the most famous folk medicines, is a necessary medicine in formulas to help other drugs reach the focal zone and maximize the effectiveness, the mechanism that promotes absorption is not clear yet. This study was carried out to investigate the absorption-promoting effects and the mechanism of benzaldehyde, a key active compound of styrax, on the diffusion rates of drugs with different oral bioavailability. Caco-2 transport experiments were used to investigate the transport rate. Molecular Dynamics Simulation analysis and fluorescence-anisotropy measurements were used to explore the underlying mechanism of absorption-promoting. Validation test in vivo was carried out to reveal the absorption-promoting effects of benzaldehyde on high hydrophilicity drugs. Our data indicated that benzaldehyde(50 µM) elevated the cumulative quantity of passively diffusion drugs with high hydrophilicity such as acyclovir and hydrochlorothiazide. MD and membrane fluidity data explained that benzaldehyde can loosen the structure of the lipid bilayer. The validation tests showed that benzaldehyde (140 mg/kg) remarkably increased the Cmax and AUC0-6 of acyclovir and hydrochlorothiazide in vivo. These present studies suggested that benzaldehyde can promote the absorption of drugs with a lower oral bioavailability through disturbing the integrity of lipid bilayer enhanced membrane permeability.

Assessment of the anti-inflammatory effects of three rhubarb anthraquinones in LPS-Stimulated RAW264.7 macrophages using a pharmacodynamic model and evaluation of the structure-activity relationships.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhubarb (Rhei Radix et Rhizoma) is a traditional Chinese medicine, has been used as a strong astringent in China to treat inflammation-related diseases, such as acute pancreatitis, acute cholecystitis, appendicitis and so on. Rhein, emodin and aloe-emodin are the important active anthraquinone in rhubarb, and are considered to be the main ingredients contributing to anti-inflammatory. AIM OF THE STUDY: Rhein, emodin and aloe-emodin, anthraquinones with the same parent structure that are found in rhubarb, have beneficial anti-inflammatory effects in vitro and in vivo. Anthraquinone derivatives also have important clinical roles. However, their pharmacodynamic differences and the structure-activity relationships associated with their anti-inflammatory properties have not been systematically explored. The present study was designed to quantify the effects of three rhubarb anthraquinones on inflammation and to explore the structure-activity relationships of these compounds. MATERIALS AND METHODS: In this study, we detected NF-κB phosphorylation, iNOS protein expression, and IL-6 and NO production in LPS-stimulated RAW264.7 cells and then calculated median effect equations and built a dynamic pharmacodynamic model to quantitatively evaluate the efficacy of these three anthraquinones. Additionally, to determine the structure-activity relationships, we investigated the physicochemical properties and molecular electrostatic potentials of the drug molecules. RESULTS: We found that rhein, emodin, and aloe-emodin exerted at least dual-target (NF-κB, iNOS) inhibition of LPS-induced inflammatory responses. Compared with rhein and emodin, aloe-emodin had a stronger anti-inflammatory effect, and its inhibition of iNOS protein expression was approximately twice that of NF-κB phosphorylation. In addition, aloe-emodin had the strongest hydrophobic effect among the three anthraquinones. CONCLUSIONS: Overall, we concluded that the receptor binding the rhubarb anthraquinones had a hydrophobic pocket. Anthraquinone molecules with stronger hydrophobic effects had higher affinity for the receptor, resulting in greater anti-inflammatory activity. These results suggest that the addition of a hydrophobic group is a potential method for structural modification to design anti-inflammatory anthraquinone derivatives with enhanced potency.

Phosphoproteomics and Proteomics Reveal Metabolism as a Key Node in LPS-Induced Acute Inflammation in RAW264.7.

To better understand the acute inflammatory mechanisms, the modulation, and to investigate the key node in predicting inflammatory diseases, high-sensitivity LC-MS/MS-based proteomics and phosphoproteomics approaches were used to identify differential proteins in RAW264.7 macrophages with lipopolysaccharide (LPS). Furthermore, differential proteins and their main biological process, as well as signaling pathways, were analyzed through bioinformatics techniques. The biological process comparison revealed 219 differential proteins and 405 differential phosphorylation proteins, including major regulatory factors of metabolism (PFKL, PGK1, GYS1, ACC, HSL, LDHA, RAB14, PRKAA1), inflammatory signaling transduction (IKKs, NF-κB, IRAK, IKBkb, PI3K, AKT), and apoptosis (MCL-1, BID, NOXA, SQSTM1). Label-free proteome demonstrated canonical inflammation signaling pathways such as the TNF signaling pathway, NF-κB signaling pathway, and NOD-like receptor signaling pathway. Meanwhile, phosphoproteome revealed new areas of acute inflammation. Phosphoproteomics profiled that glycolysis was enhanced and lipid synthesis was increased. Overall, the AMPK signaling pathway is the key regulatory part in macrophages. These revealed that the early initiation phase of acute inflammation primarily regulated the phosphoproteins of glucose metabolic pathway and lipid synthesis to generate energy and molecules, along with the enhancement of pro-inflammatory factors, and further induced apoptosis. Phosphoproteomics provides new evidence for a complex network of specific but synergistically acting mechanisms confirming that metabolism has a key role in acute inflammation.

Inhibition of eIF2α Dephosphorylation Protects Hepatocytes from Apoptosis by Alleviating ER Stress in Acute Liver Injury.

OBJECTIVES: Protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2α) is an important factor along the main pathways for endoplasmic reticulum (ER) stress-mediated apoptosis. In this study, we investigated the effects of eIF2α phosphorylation on hepatocyte apoptosis and the ER stress mechanisms in acute liver injury. METHODS: eIF2α phosphorylation and apoptosis under ER stress were monitored and measured in male BALB/c mice with acute liver injury and human hepatocyte line LO2 cells. RESULTS: Carbon tetrachloride (CCl4) administration triggered ER stress and hepatocyte apoptosis, as well as eIF2α phosphorylation in mice. Inhibition of eIF2α dephosphorylation, as the pretreatment with 4-phenylbutyric acid (chemical chaperone, ER stress inhibitor), mitigated CCl4-induced intrahepatic ER stress, apoptosis, and liver injury. In an ER stress model of LO2 cells induced by thapsigargin (disrupting ER calcium balance), inhibition of eIF2α dephosphorylation reduced ER stress and apoptosis, while PERK knockdown reduced eIF2α phosphorylation and exacerbated ER stress and apoptosis. CONCLUSIONS: eIF2α phosphorylation is one of the mechanisms employed by ER stress for restoring cellular homeostasis. Inhibition of eIF2α dephosphorylation mitigates hepatocyte apoptosis by alleviating ER stress in acute liver injuries.

Endoplasmic Reticulum Stress Increases Multidrug-resistance Protein 2 Expression and Mitigates Acute Liver Injury.

BACKGROUND: Multidrug-resistance protein (MRP) 2 is a key membrane transporter that is expressed on hepatocytes and regulated by nuclear factor kappa B (NF-κB). Interestingly, endoplasmic reticulum (ER) stress is closely associated with liver injury and the activation of NF-κB signaling. OBJECTIVE: Here, we investigated the impact of ER stress on MRP2 expression and the functional involvement of MRP2 in acute liver injury. METHODS: ER stress, MRP2 expression, and hepatocyte injury were analyzed in a carbon tetrachloride (CCl4)-induced mouse model of acute liver injury and in a thapsigargin (TG)-induced model of ER stress. RESULTS: CCl4 and TG induced significant ER stress, MRP2 protein expression and NF- κB activation in mice and LO2 cells (P < 0.05). Pretreatment with ER stress inhibitor 4- phenyl butyric acid (PBA) significantly mitigated CCl4 and TG-induced ER stress and MRP2 protein expression (P < 0.05). Moreover, pretreatment with pyrrolidine dithiocarbamic acid (PDTC; NF-κB inhibitor) significantly inhibited CCl4-induced NF-κB activation and reduced MRP2 protein expression (1±0.097 vs. 0.623±0.054; P < 0.05). Furthermore, hepatic downregulation of MRP2 expression significantly increased CCl4- induced ER stress, apoptosis, and liver injury. CONCLUSION: ER stress enhances intrahepatic MRP2 protein expression by activating NF-κB. This increase in MRP2 expression mitigates ER stress and acute liver injury.

Vanillin enhances the passive transport rate and absorption of drugs with moderate oral bioavailability in vitro and in vivo by affecting the membrane structure.

Vanillin is a popular flavoring agent in the food, tobacco, and perfume industries. In this paper, we investigated the effect of vanillin on the transport rates of drugs with different levels of permeability (acyclovir, hydrochlorothiazide, propranolol and carbamazepine) through a Caco-2 cell bidirectional transport experiment. We also explored the underlying mechanism using an in silico technique and fluorescence anisotropy measurements. The influence of vanillin on the pharmacokinetics of drugs whose transport rates were affected by vanillin in vitro was then studied in vivo. Results showed that vanillin (100 µM) increased the cumulative amount of passively transported drugs (2.1-fold of hydrochlorothiazide, 1.49-fold of propranolol, 1.35-fold of acyclovir, and 1.34-fold of carbamazepine) in vitro. Molecular dynamics simulations revealed that vanillin disordered the structure of the lipid bilayer and reduced the energy barrier of drugs across the center of the membrane. The anisotropy of TMA-DPH also decreased in Caco-2 cells after treatment with vanillin (25 and 100 µM) and indicated an increase in membrane fluidity, which was dose-dependent. An oral bioavailability study indicated that vanillin (100 mg kg-1) significantly enhanced the Cmax and AUC0-6 of hydrochlorothiazide by 1.42-fold and 1.28-fold, respectively, and slightly elevated the Cmax of propranolol. In conclusion, vanillin can significantly increase the absorption of drugs with moderate oral bioavailability in vitro and in vivo by loosening the membrane. Thus, the concurrent consumption of drugs with food containing vanillin may result in increased drug plasma concentration and pose potential health risks.

Phosphorylation of eIF2α mitigates endoplasmic reticulum stress and hepatocyte necroptosis in acute liver injury.

INTRODUCTION AND OBJECTIVES: Necroptosis and endoplasmic reticulum (ER) stress has been implicated in acute and chronic liver injury. Activated eukaryotic initiation factor 2 alpha (eIF2α) attenuates protein synthesis and relieves the load of protein folding in the ER. In this study, we aimed to analyze the impact of eIF2α phosphorylation on hepatocyte necroptosis in acute liver injury. MATERIALS AND METHODS: Male BALB/c mice were injected with tunicamycin or d-galactosamine, and LO2 cells were incubated with tunicamycin to induce acute liver injury. 4-Phenylbutyric acid (PBA) and salubrinal were used to inhibit ER stress and eIF2α dephosphorylation, respectively. We analyzed the eIF2α phosphorylation, ER stress, and hepatocyte necroptosis in mice and cells model. RESULTS: Tunicamycin or d-galactosamine significantly induced ER stress and necroptosis, as well as eIF2α phosphorylation, in mice and LO2 cells (p<0.05). ER stress aggravated tunicamycin-induced hepatocyte necroptosis in mice and LO2 cells (p<0.05). Elevated eIF2α phosphorylation significantly mitigated hepatocyte ER stress (p<0.05) and hepatocyte necroptosis in mice (34.37±3.39% vs 22.53±2.18%; p<0.05) and LO2 cells (1±0.11 vs 0.33±0.05; p<0.05). Interestingly, tumor necrosis factor receptor (TNFR) 1 protein levels were not completely synchronized with necroptosis. TNFR1 expression was reduced in d-galactosamine-treated mice (p<0.05) and cells incubated with tunicamycin for 12 and 24h (p<0.05). ER stress partially restored TNFR1 expression and increased necroptosis in tunicamycin-incubated cells (p<0.05). CONCLUSIONS: These results imply that ER stress can mediate hepatocyte necroptosis independent of TNFR1 signaling and elevated eIF2α phosphorylation can mitigate ER stress during acute liver injury.

Coptisine from Coptis chinensis blocks NLRP3 inflammasome activation by inhibiting caspase-1.

Inflammasome mediates the activation of caspase-1, which promotes the secretion of proinflammatory cytokines. In this work, we aimed to investigate whether natural compounds from a Traditional Chinese Medicine prescription called San-Huang-Xie-Xin-Tang exert its clinical efficacy by inhibiting inflammasome activation and the underlying mechanism. The inhibitory effects of compounds on caspase-1 were evaluated in recombinant expressed caspase-1 protein and macrophages. Molecular docking was conducted to examine the interaction between compounds and caspase-1. The effects of the compounds on pro-inflammatory cytokines were examined by enzyme-linked immunosorbent assay. The mechanism of the compounds on nucleotide oligomerization domain (NOD)-like receptor protein-3 (NLRP3) inflammasome activation was investigated in macrophages. The anti-inflammasome effects of compounds were examined in mice stimulated by lipopolysaccharide (LPS) and monosodium urate crystal (MSU). Coptisine was the most potent inhibitor of caspase-1 in the San-Huang-Xie-Xin-Tang prescription. Coptisine adopted a favorable conformation at the active site of caspase-1. Coptisine significantly attenuated mature interleukin (IL)-1ß secretion in RAW264.7 macrophages stimulated with LPS plus ATP, nigericin, or MSU, by blocking caspase-1 activation. Coptisine not only prevented NLRP3 inflammasome assembly by affecting the binding between pro-caspase-1 and apoptosis-associated speck-like protein containing a CARD, but also inhibited inflammasome priming by decreasing NLRP3 expression through inactivation of the nuclear factor-κB pathway. Moreover, coptisine prevented LPS-mediated IL-1ß production and MSU-mediated mice paw edema by blocking NLRP3 inflammasome activation in vivo. Coptisine blocks NLRP3 inflammasome activation by inhibiting caspase-1 and may be useful for treating NLRP3 inflammasome-involved gouty arthritis.

Pharmacokinetic-Pharmacodynamic Modeling for Coptisine Challenge of Inflammation in LPS-Stimulated Rats.

Pro-inflammatory factors are important indicators for assessing inflammation severity and drug efficacy. Coptisine has been reported to inhibit LPS-induced TNF-α and NO production. In this study, we aim to build a pharmacokinetic-pharmacodynamic model to quantify the coptisine time course and potency of its anti-inflammatory effect in LPS-stimulated rats. The plasma and lung coptisine concentrations, plasma and lung TNF-α concentrations, plasma NO concentration, and lung iNOS expression were measured in LPS-stimulated rats after intravenous injection of three coptisine doses. The coptisine disposition kinetics were described by a two-compartment model. The coptisine distribution process from the plasma to the lung was described by first-order dynamics. The dynamics of plasma TNF-α generation and elimination followed zero-order kinetics and the Michaelis-Menten equation. A first-order kinetic model described the TNF-α diffusion process from the plasma to the lung. A precursor-pool indirect response model was used to describe the iNOS and NO generation induced by TNF-α. The inhibition rates of TNF-α production by coptisine (54.73%, 26.49%, and 13.25%) calculated from the simulation model were close to the decline rates of the plasma TNF-α AUC (57.27%, 40.33%, and 24.98%, respectively). Coptisine suppressed plasma TNF-α generation in a linear manner, resulting in a cascading reduction of iNOS and NO. The early term TNF-α response to stimulation is a key factor in the subsequent inflammatory cascade. In conclusion, this comprehensive PK-PD model provided a rational explanation for the interlocking relationship among TNF-α, iNOS and NO production triggered by LPS and a quantitative evaluation method for inhibition of TNF-α production by coptisine.

Inhibiting alpha subunit of eukaryotic initiation factor 2 dephosphorylation protects injured hepatocytes and reduces hepatocyte proliferation in acute liver injury.

AIM: To investigate the impact of alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation on liver regeneration. METHODS: Male BALB/c mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver injury. Human hepatocyte LO2 cells were incubated with thapsigargin to induce endoplasmic reticulum (ER) stress. Salubrinal, integrated stress response inhibitor (ISRIB), and DnaJC3 overexpression were used to alter eIF2α phosphorylation levels. RESULTS: CCl4 administration induced significant ER stress and eIF2α phosphorylation, and increased hepatocyte proliferation proportionally to the extent of injury. Inhibiting eIF2α dephosphorylation with salubrinal pretreatment significantly mitigated liver injury and hepatocyte proliferation. In LO2 cells, thapsigargin induced significant eIF2α phosphorylation and inhibited proliferation. Inhibiting eIF2α dephosphorylation partly restored cell proliferation during ER stress. CONCLUSIONS: In acute liver injury, inhibiting eIF2α dephosphorylation protects injured hepatocytes and reduces hepatocyte proliferation.

Semi-Mechanism-Based Pharmacodynamic Model for the Anti-Inflammatory Effect of Baicalein in LPS-Stimulated RAW264.7 Macrophages.

Monitoring of the inhibition of TNF-α, IL-6, iNOS, and NO is used to effectively evaluate anti-inflammatory drugs. Baicalein was found to have good anti-inflammatory activities, but its detailed cellular pharmacodynamic events have not been expatiated by any other study. The inflammatory mediators, including TNF-α, IL-6, iNOS, and NO production in RAW264.7 macrophage induced by LPS, were measured. It was found that these data showed a sequential pattern on time and based on these points a cellular pharmacodynamic model was developed and tested. TNF-α and IL-6 were quantified by ELISA, NO was detected by Griess and iNOS expression was measured by Western blot. The pharmacodynamic model was developed using a NLME modeling program Monolix® 2016R1.The results showed that baicalein quickly suppressed release of TNF-α in a concentration-dependent manner, and consequently causing the diminution of IL-6 and iNOS/NO. The pharmacodynamic model simulation successfully described the experimental data, supporting the hypothesis that IL-6 and iNOS /NO release after LPS stimulation is mediated by TNF-α rather than LPS directly. The pharmacodynamic model allowed a well understanding of the cellular pharmacodynamic mechanism of baicalein in the treatment of inflammatory diseases.

San-Huang-Xie-Xin-Tang Constituents Exert Drug-Drug Interaction of Mutual Reinforcement at Both Pharmacodynamics and Pharmacokinetic Level: A Review.

Inflammatory disorders underlie varieties of human diseases. San-Huang-Xie-xin-Tang (SHXXT), composed with Rhizoma Rhei (Rheum palmatum L.), Rhizoma Coptidis (Coptis chinensis Franch), and Radix Scutellaria (Scutellaria baicalensis Georgi), is a famous formula which has been widely used in the fight against inflammatory abnormalities. Mutual reinforcement is one of the basic theories of traditional Chinese medicine. Here this article reviewed and analyzed the recent research on (1) How the main constituents of SHXXT impact on inflammation-associated signaling pathway molecules. (2) The interaction between the main constituents and efflux pumps or intestinal transporters. The goal of this work was to, (1) Provide evidence to support the theory of mutual reinforcement. (2) Clarify the key targets of SHXXT and suggest which targets need further investigation. (3) Give advice for the clinical use of SHXXT to elevated the absorption of main constituents and eventually promote oral bioavailability. We search literatures in scientific databases with key words of "each main SHXXT constituent," in combination with "each main inflammatory pathway target molecule" or each main intestinal transporter, respectively. We report the effect of five main constituents on target molecules which lies in three main inflammatory signaling pathways, we as well investigate the interaction between constituents and intestinal transporter. We conclude, (1) The synergistic effect of constituents at both levels confirm the mutual reinforcement theory of TCM as it is proven in this work. (2) The effect of main constituents on downstream targets in nuclear need more further investigation. (3) Drug elevating the absorption of rhein, berberine and baicalein can be employed to promote oral bioavailability of SHXXT.

Suppression of murine melanoma growth by a vaccine of attenuated Salmonella carrying heat shock protein 70 and Herpes simplex virus-thymidine kinase genes.

Attenuated Salmonella can invade tumor cells and acts as a eukaryotic expression vector for gene propagation. We constructed a bi-gene, eukaryotic co-expression DNA vaccine of Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and Herpes simplex virus-thymidine kinase (HSV-tk) and used attenuated Salmonella as a vector to treat murine melanoma. In vitro, recombinant Salmonella can carry plasmid stably and can invade into the cytoplasm of B16 tumor cells expressing the protein of the mtHSP70/HSV-tk gene by Western blot assay. In vivo, after the recombinant Salmonella was injected into tumors, the HSV-tk precursor drug ganciclovir (GCV) was administered to start the HSV-tk killing of tumor cells. We found that the mtHSP70/HSV-tk recombinant bacteria can raise CD8+ T lymphocytes in peripheral blood by flow cytometry and in tumor tissues by immunofluorescence detection, increase IFN­Î³ contents in tumor tissue by ELISA and significantly suppress tumor growth.

Bone marrow mesenchymal stem cells reduce the antitumor activity of cytokine-induced killer/natural killer cells in K562 NOD/SCID mice.

Adoptive cellular immunotherapy is an important treatment to eliminate residual tumor cells after hematopoietic stem-cell transplantation. Bone marrow mesenchymal stem cells (MSC) have previously been shown to exert immunoregulation functions, including inhibition of proliferation and killing activities of T cells and natural killer (NK) cells in vitro and reduction of the graft-versus-host disease. MSC can survive in vivo for a long period of time, the influence of MSC on the antitumor activity of subsequently infused immune killer cells is not clear. The aim of this study was to investigate the influences of MSC infused via different paths and at different times on the antitumor activities of cytokine-induced killer (CIK)/NK cells derived from umbilical cord blood in K562 NOD/SCID mice. The potential interaction mechanisms of MSC and CIK/NK cells infused through different paths using different intervals in vivo were subsequently explored. The results show that the antitumor activities of CIK/NK cells was inhibited by MSC when injected via the same path (tail vein), and the suppressive effect of MSC on CIK/NK cells were less pronounced when they were injected separately through different paths. There were no effects of MSC on the antitumor activities of CIK/NK cells if the MSC and CIK/NK cells were injected with a 48-h interval. Moreover, the suppressive effect continuous, even if MSC were infused 48 h earlier than CIK/NK cells. It suggests that pre-injected MSC can reduce the antitumor activities of CIK/NK cells in vivo. The probable mechanisms are that MSC and CIK/NK cells might have a greater opportunity to meet and interact if they are injected simultaneously via the same path. The suppression of MSC on CIK/NK cells in vivo mainly takes place in the reticuloendothelial system, including the lung and the liver.

[Distribution and interaction of bone marrow mesenchymal stem cells and cord blood CIK/NK cells infused via different ways at different time periods in NOD/SCID mice].

The study was aimed to explore the distribution and interaction mechanism of human bone marrow mesenchymal stem cells (MSC) and cord blood cytokine-induced killer (CIK)/natural killer (NK) cells infused via different ways at different times in NOD/SCID mice. 5 microl 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) dye(red) was added in suspension of MSC per ml, and 1 microl carboxyfluorescein diacetate, succinimidyl ester(CFDA SE) dye(green) was added in suspension of CIK/NK cells per ml. The amounts of MSC and CIK/NK cells infused in each 6 NOD/SCID mouse were 1 x 10(6) (0.1 ml) and 1 x 10(7) (0.1 ml) respectively. All mice were divided into 4 groups, each group consisted of 6 mice. Group A: MSC (intravenous infusion, iv) + CIK/NK cells (iv) at the same time, group B: MSC (iv) + CIK/NK cells (iv) at 48 hours after infusion of MSC; group C: MSC (intramedullary infusion, im) + CIK/NK cells (iv) at the same time; group D: MSC (im) + CIK/NK cells (iv) at 48 hours after infusion of MSC. 3 NOD/SCID mice were sacrificed per batch at 24 hours and 48 hours after infused CIK/NK cells. Frozen sections of liver, spleen, lung and kidney were prepared, and then followed by counting the amounts of red and green fluorescence cells under fluorescence microscope, and calculating the ratio of MSC to CIK/NK cells for reflecting the interaction of MSC and CIK/NK cells in mice, and for showing the suppressive intensity of MSCs on CIK/NK cells. The results showed that the sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen of group A and B were higher than that in group C and D at 24 hours and 48 hours respectively after infusing CIK/NK cells. The sum of average ratios of MSC to CIK/NK cells in group A was slightly higher than that in group B at 24 hours and 48 hours after infusing CIK/NK cells, but there was no significant difference between them. The sum of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group C was slightly lower than that in group D at 24 hours after infusing CIK/NK cells, but reversed at 48 hours later and there was no significant difference between them. The sums of average ratios of MSC to CIK/NK cells in lung, liver and spleen in group A, B, C and D were all higher than those in kidney at 24 and 48 hours respectively after infusing CIK/NK cells. It is concluded, the MSC and CIK/NK cells may interact if they are infused via the same way and at the same time, the location where the suppression of MSC on CIK/NK cells occur in vivo may be reticulo-endothelial systems in lungs and livers.