p-coumaric acid (p-CA), a common plant phenolic acid with multiple bioactivities, has a lipid-lowering effect. As a dietary polyphenol, its low toxicity, with the advantages of prophylactic and long-term administration, makes it a potential drug for prophylaxis and the treatment of nonalcoholic fatty liver disease (NAFLD). However, the mechanism by which it regulates lipid metabolism is still unclear. In this study, we studied the effect of p-CA on the down-regulation of accumulated lipids in vivo and in vitro. p-CA increased a number of lipase expressions, including hormone-sensitive lipase (HSL), monoacylglycerol lipase (MGL) and hepatic triglyceride lipase (HTGL), as well as the expression of genes related to fatty acid oxidation, including long-chain fatty acyl-CoA synthetase 1 (ACSL1), carnitine palmitoyltransferase-1 (CPT1), by activating peroxisome proliferator-activated receptor α, and γ (PPARα and γ). Furthermore, p-CA promoted adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and enhanced the expression of the mammalian suppressor of Sec4 (MSS4), a critical protein that can inhibit lipid droplet growth. Thus, p-CA can decrease lipid accumulation and inhibit lipid droplet fusion, which are correlated with the enhancement of liver lipases and genes related to fatty acid oxidation as an activator of PPARs. Therefore, p-CA is capable of regulating lipid metabolism and is a potential therapeutic drug or health care product for hyperlipidemia and fatty liver.

Berberine (BBR) has a variety of pharmacological activities.Studies have reported that BBR not only reduces heat stress-induced fever but also inhibits lower body temperatures due to cold stress.Heat stress can be reduced via BBR treatment,which antagonizes HSP70-TNFα to regulate the body temperature alteration.In cold stress,however,the molecular mechanism of BBR-induced inhibition of hypothermia remains unclear.Therefore,we studied whether BBR promoted uncoupling protein 1 (UCP1,a crucial protein of thermogenesis) expression and its mechanism under cold stress.Wild type mice and Ucp1-/ mice were used for the in vivo experiments,and primary brown adipocytes and brown adipocytes HIB-1B were used for the in vitro studies.The cold stress was set at 4 ℃.The results showed that at 4 ℃C,the body temperature of mice was decreased.BBR effectively inhibited this hypothermia.Simultaneously,Ucp1 expression in brown adipose tissue (BAT) cells was significantly increased,and BBR promoted Ucp1 expression.However,in Ucp1-knockout mice,the effect of BBR on hypothermia disappeared during cold stress,indicating that the main target for BBR regulation of body temperature was Ucp1.Further studies showed that the transcriptional response element NFE2 (nuclear factor erythroid-derived 2) in the upstream of the Ucp1 promoter region contributed to the positive regulatory role on Ucp1 expression at lower temperature.BBR could bind to the sequence of NFE2 response element in a temperature-dependent manner.Increased affinity of BBR binding to NFE2 response element in cold stress significantly strengthened and enhanced the expression of Ucp1.This work was important for understanding the role of BBR on thermogenesis in BAT,body temperature regulation and temperature tolerance under cold conditions.

Transient Receptor Potential Melastatin-8 (TRPM8) reportedly plays a fundamental role in a variety of processes including cold sensation, thermoregulation, pain transduction and tumorigenesis. However, the role of TRPM8 in inflammation under cold conditions is not well known. Since cooling allows the convergence of primary injury and injury-induced inflammation, we hypothesized that the mechanism of the protective effects of cooling might be related to TRPM8. We therefore investigated the involvement of TRPM8 activation in the regulation of inflammatory cytokines. The results showed that TRPM8 expression in the mouse hypothalamus was upregulated when the ambient temperature decreased; simultaneously, tumor necrosis factor-alpha (TNFα) was downregulated. The inhibitory effect of TRPM8 on TNFα was mediated by nuclear factor kappa B (NFκB). Specifically, cold stress stimulated the expression of TRPM8, which promoted the interaction of TRPM8 and NFκB, thereby suppressing NFκB nuclear localization. This suppression consequently led to the inhibition of TNFα gene transcription. The present data suggest a possible theoretical foundation for the anti-inflammatory role of TRPM8 activation, providing an experimental basis that could contribute to the advancement of cooling therapy for trauma patients.

After being studied for approximately a century, berberine (BBR) has been found to act on various targets and pathways. A great challenge in the pharmacological analysis of BBR at present is to identify which target(s) plays a decisive role. In the study described herein, a rescue experiment was designed to show the important role of mitochondria in BBR activity. A toxic dose of BBR was applied to inhibit cell proliferation and mitochondrial activity, then α-ketobutyrate (AKB), an analogue of pyruvate that serves only as an electron receptor of NADH, was proven to partially restore cell proliferation. However, mitochondrial morphology damage and TCA cycle suppression were not recovered by AKB. As the AKB just help to regenerate NAD+, which is make up for part function of mitochondrial, the recovered cell proliferation stands for the contribution of mitochondria to the activity of BBR. Our results also indicate that BBR suppresses tumour growth and reduces energy charge and mitochondrial DNA (mtDNA) copy number in a HepG2 xenograft model. In summary, our study suggests that mitochondria play an important role in BBR activity regarding tumour cell proliferation and metabolism.

Pomegranate leaf (PGL) has a definite role in regulating lipid metabolism. However, pharmacokinetic results show the main active ingredient, ellagic acid, in PGL has lower oral bioavailability, suggesting that the lipid-lowering effect of PGL may act through inhibiting lipid absorption in the small intestine. Our results demonstrated that pomegranate leaf and its main active ingredients (i.e., ellagic acid, gallic acid, pyrogallic acid and tannic acid) were capable of inhibiting pancreatic lipase activity in vitro. In computational molecular docking, the four ingredients had good affinity for pancreatic lipase. Acute lipid overload experiments showed that a large dosage of PGL significantly reduced serum total cholesterol (TG) and triglycerides (TC) levels in addition to inhibiting intestinal lipase activity, which demonstrated that PGL could inhibit lipase activity and reduce the absorption of lipids. We also found that PGL could reverse the reduced tight-junction protein expression due to intestinal lipid overload, promote Occludin and Claudin4 expression in the small intestine, and enhance the intestinal mucosal barrier. In conclusion, we demonstrated that PGL can inhibit lipid absorption and reduce blood TG and TC by targeting pancreatic lipase, promoting tight-junction protein expression and thereby preventing intestinal mucosa damage from an overload of lipids in the intestine.

OBJECTIVE:To explore the anti-nociceptive effect of patchouli alcohol (PA), the essential oil isolated from Pogostemon cablin (Blanco) Bent, and determine the mechanism in molecular levels.METHODS:The acetic acid-induced writhing test and formalin-induced plantar injection test in mice were employed to confirm the effect in vivo. Intracellular calcium ion was imaged to verify PA on mu-opioid receptor (MOR). Cyclooxygenase 2 (COX2) and MOR of mouse brain were expressed for determination of PA's target. Cellular experiments were carried out to find out COX2 and MOR expression induced by PA.RESULTS:PA significantly reduced latency period of visceral pain and writhing induced by acetic acid saline solution (P<0.01) and allodynia after intra-plantar formalin (P<0.01) in mice. PA also up-regulated COX2 mRNA and protein (P<0.05) with a down-regulation of MOR (P<0.05) both in in vivo and in vitro experiments, which devote to the analgesic effect of PA. A decrease in the intracellular calcium level (P<0.05) induced by PA may play an important role in its anti-nociceptive effect. PA showed the characters of enhancing the MOR expression and reducing the intracellular calcium ion similar to opioid effect.CONCLUSIONS:Both COX2 and MOR are involved in the mechanism of PA's anti-nociceptive effect, and the up-regulation of the receptor expression and the inhibition of intracellular calcium are a new perspective to PA's effect on MOR.

Berberine (BBR) is a natural small molecule with various pharmacological activities and biological targets.BBR has been shown to inhibit mRNA decay in our previous studies,which is associated with its high binding affinity to the poly-adenine (poly A) tail at the 3'end of mRNA.However,the exact mechanism remains unknown.In this research,we discovered that deficiency of cytoplasmic poly A binding protein (PABP),which protects mRNA from nucleolytic attack as a poly A-PABP complex,led to the loss of BBR's effect on mRNA decay inhibition.We also demonstrated using fluorescence spectroscopy,RNA-EMSA (RNA-electrophoretic mobility shift assay) in vitro,and RIP (RNA immunoprecipitation) that BBR could significantly promote PABP binding to poly A.We might conclude that BBR could stabilize mRNA by enhancing the interaction between poly A and PABP.In addition,the HMBC (1H detected heteronuclear multiple bond correlation) studies demonstrated that BBR could bind to AMP,a monomer of poly A,directly and specifically.Further evidence of molecular docking suggested that BBR might act as a linker to stabilize the poly A-PABP,and elongate the half-life of mRNAs.This demonstrates that BBR might affect protein translation initiation and up-regulate protein expression.

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.

Brazilein is reported to have immunosuppressive effect on cardiovascular and cerebral-vascular diseases. The essential roles of innate immunity in cerebral ischemia are increasingly identified, but no studies concerning the influence of brazilein on the innate immunity receptors have been reported. The present study was designed to investigate the regulation of NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) by brazilein for its protection of neuron in cerebral ischemia in vivo and oxygen-glucose deprivation in vitro. The results showed that brazilein could reverse the elevated expression of NOD2 and TNFα (tumor necrosis factor alpha) elicited by cerebral ischemia and reperfusion. This reduction could also be detected in normal mice and C17.2 cells, indicating that this suppressive effect of brazilein was correlated with NOD2. The results from GFP reporter plasmid assay suggested brazilein inhibited NOD2 gene transcription. In conclusion, brazilein could attenuate NOD2 and TNFα expression in cerebral ischemia and NOD2 may be one possible target of brazilein for its immune suppressive effect in neuro-inflammation.

本文以中药小分子为例对小分子药物的跨膜转运研究进展及方法进行了综述,并结合作者研究成果,对小分子药物跨膜转运与作用靶点进行了分析和探讨。目前,研究小分子转运的检测方法有HPLC、LC/MS,还可以借助其荧光成像利用共聚焦技术进行观测。在研究过程中,确定小分子的转运方式的同时,也要探讨其外排方式,并要确定其有无代谢及其可能的代谢产物。对小分子药物的细胞跨膜转运研究将有助于确定小分子作用的靶点,有助于对其药理作用的探讨和阐释。

A variety of pharmacological effects of berberine (BBR) are constantly being discovered with the deepening of BBR research. What followed is how to rationally use the drug according to these new pharmacological effects. Because of some cardiac toxicity and poor oral absorption, conflicts may arise between improving the bioavailability and controlling the toxicity of BBR. Meanwhile some new therapeutic uses of BBR, such as hypolipidemia, hypoglycemia as well as prevention and treatment of neurodegenerative diseases, need long-termoral administration, thereby may lead to alteration of intestinal flora and potentially affect body's other physiological functions. Based on the stated targets of BBR and related pharmaceutical properties, comprehensive analysis of these issues was conducted in this study. Some suggestions were presented below：the effect of long-term oral administration on body function, especially the intestinal flora, needs to be further investigated; risks shall be considered in changing the composition of the formulation to improve the absorption rate of oral administration; for the medication with higher concentration demand (such as anti-cancer), targeted drug-delivery is worthy to be considered.

Angelica dahurica (A. dahurica) is a traditional Chinese medicinal plant being used in clinical practice. The present study demonstrated that A. dahurica could reduce white-fat weight in high-fat-diet hyperlipidemic mice, decrease total cholesterol and triglyceride concentrations in the livers of both high-fat-diet and Triton WR1339 induced hyperlipidemic mice, and enhance the total hepatic lipase activities of them. These findings were further supported by the results derived from the experiments with HepG2 cells in vitro. In addition, the proteins related to lipids metabolism were investigated using LC-MS/MS, indicating that genes of lipid metabolism and lipid transport were regulated by A. dhurica. The results from LC-MS/MS were further conformed by Western blot and real time PCR assays. A. dahurica could down-regulate the expression of catalase (CAT) and sterol carrier protein2 (SCP2) and up-regulate the expression of lipid metabolism related genes-lipase member C (LIPC) and peroxisome proliferator-activated receptor gamma (PPARγ). In the Triton WR1339 mouse liver and HepG2 cells in vitro, A. dahurica was able to increase the expression of LIPC and PPARγ, confirming the results from in vivo experiments. Imperatorin showed the same activity as A. dahurica, suggesting it was one of the major active ingredients of the herb. In conclusion, our work represented a first investigation demonstrating that A. dahurica was able to regulate lipid metabolism and could be developed as a novel approach to fighting against fatty liver and obesity.

This study was aimed to observe the effect ofpomegranate leaf tennis and Ananas coumosus leaf phenols on the growth of lactating mice through breast milk. Intragastric administration of different doses of drug was given to maternal mice. The general condition of newborn mice, body weight, tail length and organ coefficient of liver-related factors regulating the expression of lipid metabolism were observed. The results showed that both pomegranate leaf tennis and A. coumosus leaf phenols can obviously affect the growth and development of newborn mice through breast milk. And the effect of pomegranate leaf tennis was stronger than A. coumosus leaf phenols. Meanwhile, it obviously downregulated the expression of liver-related lipid metabolism enzymes in newborn mice. It was concluded that pomegranate leaf tennis and A. coumosus leaf phenols can affect the growth and development of newborn mice through breast milk. Its effect was related to the influence of lipid metabolism enzyme in the liver. Attention should be paid on taking this medication during lactation. Its clinical significance still required in-depth study.

This study was aimed to observe effect of pomegranate leaf tannin and ellagic acid on primary rat adipocyte differentiation and lipid metabolism-related factor expression. Primary rat preadipocyte was in vitro cultured to observe the effect of pomegranate leaf tannin and ellagic acid on lipid fat cells as well as mRNA expression of its related factor. The results showed that pomegranate leaf tannin and ellagic acid had obvious inhibition effect on fat formation in fat cells. It had certain inhibition effect on activities of lipoprotein lipase (LPL) and glucose-3-phosphate dehydrogenase (GPDH). It promoted fat decomposition and reduced intracellular lipid content. It upregulated PPARγ and fatty acid-binding protein (aP2). It downregulated obese (ob) gene level. It was concluded that pomegranate leaf tannin can inhibit fat generation of fat cells and promote fat metabolism. Ellagic acid was its main active ingredient, which had the same effect as pomegranate leaf tannin.

The purpose of the present study is to confirm the protective effect of berberine (BBR) on gastrointestinal injury caused by acute heavy alcohol exposure, an effect that has not been reported previously. Our research details how BBR protects against gastrointestinal injuries from acute alcohol exposure using both in vivo and in vitro experiments. Acute high alcohol concentrations lead to obvious damage to the gastrointestinal mucosa, resulting in necrosis of the intestinal mucosa. Oral administration of BBR was able to significantly reduce this alcohol-induced damage, inhibit increases of alcohol-induced TNFα and IL-1β expression in gastrointestinal mucosa as well as their upstream signals TLR2 and TLR4, and regulate cytokines that modulate tight junctions. Alcohol consumption is a popular human social behavior worldwide, and the present study reports a comprehensive mechanism by which BBR protects against gastrointestinal injuries from alcohol stress, providing people with a novel application of BBR.

Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine's pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies.

Brazilein, a natural small molecule, shows a variety of pharmacological activities, especially on nervous system and immune system. As a potential multifunctional drug, we studied the distribution and the transport behavior and metabolic behavior of brazilein in vivo and in vitro. Brazilein was found to be able to distribute in the mouse brain and transport into neural cells. A metabolite was found in the brain and in the cells. Positive and negative mode-MS/MS and Q-TOF were used to identify the metabolite. MS/MS fragmentation mechanisms showed the methylation occurred at the 10-hydroxyl of brazilein (10-O-methylbrazilein). Further, catechol-O- methyltransferase (COMT) was confirmed as a crucial enzyme correlated with the methylated metabolite generation by molecular docking and pharmacological experiment.

Berberine is one kind of isoquinoline alkaloid with anti-apoptotic effects on the neurons suffering ischemia. To address the explanation for these activities, the berberine-induced cell cycle arrest during neurons suffering ischemia/reperfusion had been studied in the present study. According to the in vitro neurons with oxygen-glucose deprivation and in vivo ICR mice with cerebral ischemia/reperfusion, it was found that berberine could protect the mRNA of retinoblastoma (Rb) from degradation through its function on the poly(A) tail. The prolonged half-life of retinoblastoma 1 (gene of Rb, RB1) mRNA level secures the protein level of retinoblastoma, which facilitates cell cycle arrest of neurons in the process of ischemia/reperfusion and subsequently avoids cells entering in the apoptotic process. The poly(A) tail of RB1 mRNA, as a newly identified target of berberine, could help people focus on the interaction between berberine and mRNA to further understand the biological activities and functions of berberine.

This article was aimed to study the general reproductive toxicity in mice in order to give a better evalua-tion of the medicinal plant of pineapple leaves (A nanas comasus L). Adult male and female mice were orally admin-istered with pineapple leaves. And then, each of the male and female mice was put together in one cage for mating. The mating success females were fed continuously. The experimental observation was conducted in pregnancy, fetal development, as well as the offspring of mice. The results showed that in addition to a large dose of pineapple leaves (4 g·kg-1) of the parental male rats having a lower body weight, pineapple leaves did not significantly affect on other parameters. There were no significant effects on pregnant mice and their offspring of mice. It was concluded that the pineapple leaves did not influence the general reproductive function of mice apparently.