Objective To explore the effects and mechanism of ursolic acid (UA) on cholesterol metabolism in human hepatocellular carcinoma HepG2 and mouse hepatocyte AML-12. Methods HepG2 and AML-12 cells were treated with different concentrations of UA (0,10,20,40 μmol·L-1)for 24 h, then the mRNA and protein expression of cholesterol 7alpha-hydroxylase (CYP7A1) and intracellular cholesterol level was detected by RT-PCR、Western blotting and enzymatic method, respectively.Results Compared with 0 μmol·L-1 UA, 20 μmol·L-1 and 40 μmol·L-1 UA significantly increased the expressions of CYP7A1 mRNA and protein(P<0.05), and decreased intracellular cholesterol level in HepG2 and AML-12 cells (P<0.05). Conclusion A certain concentration of UA can reduce the level of cholesterol in HepG2 and AML-12 cells. CYP7A1 may be involved in the regulation process.
1.3.4 RT-PCR检测CYP7A1 mRNA表达 应用TRIzol试剂提取细胞总RNA,然后采高容量cDNA反转录试剂盒合成cDNA。RT-PCR引物采用Primer Premier 5版软件设计,引物序列见表1。应用SYBR Green RT-PCR 技术检测CYP7A1 mRNA表达水平。以β-actin作为内参对照。整个体系在RT-PCR仪中进行扩增,然后作相对定量分析。
表1
Tab.1
表1
表1
CYP7A1和β-actin的引物序列
Tab.1
Primer sequences of CYP7A1 and β-actin
基因名称
引物序列
扩增 长度/bp
CYP7A1 (人)
Sense: 5'-AGAAATCTACCCAGACCCTT-3'
Anti-sense: 5'-TTGATTTCGTGGATAGCGAA-3'
171
CYP7A1 (鼠)
Sense: 5'-TTCTTTGATCTGGGGGATTG-3'
Anti-sense: 5'-GTTTGCTTTGCTTTGCTCTT-3'
176
β-actin (人)
Sense: 5'-ACTATCGGCAATGAGCG-3'
Anti-sense: 5'-GAGCCAGGGCAGTAATCT-3'
220
β-actin (鼠)
Sense: 5'-GACGGCCAGGTCATCACTAT-3'
Anti-sense: 5'-CGGATGTCAACGTCACACTT-3'
140
表1
CYP7A1和β-actin的引物序列
Tab.1
Primer sequences of CYP7A1 and β-actin
1.3.5 Western blotting检测CYP7A1表达 HepG2及AML-12细胞分别给予终浓度为0,10,20和40 μmol·L-1的熊果酸处理24 h,提取总蛋白,并测定蛋白浓度。10%蛋白质聚丙烯酰胺分离,应用电转移方法,将蛋白转至聚偏氟乙烯膜上,5%牛血清清蛋白封闭30 min,加入一抗,4 ℃振荡过夜。用含1%聚山梨酯的TBS(TBST)洗涤3次后,加入碱性磷酸酶标记的二抗,室温孵育1 h,TBST洗涤3次后,加入显色剂,显色后拍摄图像保存,并以β-actin作为内参对照进行分析。
Fig.1
Effects of ursolic acid on the protein expression of CYP7A1 in HepG2 and AML-12 cells (x¯±s,n=4) Compared with 0 μmol·L-1 ursolic acid group,*1P<0.05
SHISHODIAS, MAJUMDARS, BANERJEES, et al.Ursolic acid inhibits nuclear factor-kappa B activation induced by carcinogenic agents through suppression of Ikappa B alpha kinase and p65 phosphorylation: correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1[J]. Cancer Res, 2003, 63(15):4375-4383.
Abstract The process of tumorigenesis requires cellular transformation, hyperproliferation, invasion, angiogenesis, and metastasis. Several genes that mediate these processes are regulated by the transcription factor nuclear factor-kappaB (NF-kappaB). The latter is activated by various carcinogens, inflammatory agents, and tumor promoters. Thus, agents that can suppress NF-kappaB activation have the potential to suppress carcinogenesis. Ursolic acid, a pentacyclic triterpene acid, has been shown to suppress the expression of several genes associated with tumorigenesis, but whether ursolic acid mediates its effects through suppression of NF-kappaB is not understood. In the study described in the present report, we found that ursolic acid suppressed NF-kappaB activation induced by various carcinogens including tumor necrosis factor (TNF), phorbol ester, okadaic acid, H(2)O(2), and cigarette smoke. These effects were not cell type specific. Ursolic acid inhibited DNA binding of NF-kappaB consisting of p50 and p65. Ursolic acid inhibited IkappaBalpha degradation, IkappaBalpha phosphorylation, IkappaBalpha kinase activation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Ursolic acid also inhibited NF-kappaB-dependent reporter gene expression activated by TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor, NF-kappaB-inducing kinase, IkappaBalpha kinase, and p65. The inhibition of NF-kappaB activation correlated with suppression of NF-kappaB-dependent cyclin D1, cyclooxygenase 2, and matrix metalloproteinase 9 expression. Thus, overall, our results indicate that ursolic acid inhibits IkappaBalpha kinase and p65 phosphorylation, leading to the suppression of NF-kappaB activation induced by various carcinogens. These actions of ursolic acid may mediate its antitumorigenic and chemosensitizing effects.
TSAI SJ, YIN MC.Antioxidative and anti-inflammatory protection of oleanolic acid and ursolic acid in PC12 cells[J]. J Food Sci, 2008, 73(7):174-178.
ABSTRACT:鈥 PC12 cells were used to examine the antioxidative and anti-inflammatory effects of oleanolic acid (OA) and ursolic acid (UA). PC12 cells were pretreated with OA or UA at 20 and 40 M and followed by exposure of hydrogen peroxide (HO) or 1-methyl-4-phenylpyridinium ion (MPP) to induce cell injury. Results showed that HO- or MPP-treatment significantly decreased cell viability and increased lactate dehydrogenase (LDH) release (< 0.05). The pretreatment from OA or UA significantly and concentration-dependently reduced subsequent HO- or MPP-induced cell death and LDH release (< 0.05). Either HO- or MPP-treatment significantly increased malonyldialdehyde (MDA) formation, decreased glutathione (GSH) content, and diminished glutathione peroxidase (GPX), catalase, and superoxide dismutase (SOD) activities (< 0.05). The pretreatment from OA or UA significantly retained GSH, and reversed HO- and MPP-induced impairment in catalase and SOD activities (< 0.05), and decreased MDA formation (< 0.05). Either HO- or MPP-treatment significantly elevated interleukin-6 (IL-6) and tumor necrosis factor (TNF)-伪 levels (< 0.05). The pretreatments from OA or UA significantly attenuated subsequent HO- or MPP-induced release of IL-6 and TNF-伪 (< 0.05). Based on the observed antioxidative and anti-inflammatory activities from OA and UA, these 2 compounds were potent agents against neurodegenerative disorder.
BAGLINI, MITAINE-OFFER A C, NOUR M, et al.A review of natural and modified betulinic, ursolic and echinocystic acid derivatives as potential antitumor and anti-HIV agents[J]. Mini Rev Med Chem, 2003, 3(6):525-539.
The aim of this review is to update current knownledge on the betulinic, ursolic and echinocystic acids and their natural and semisynthetic analogs, focussing on their cytotoxic and anti-HIV activities. Then, the last results of the authors' team on unusual semisynthetic derivatives of these triterpenoids will be presented in order to establish structure / activity relationships.
KIMJ, JANG DS, KIMH, et al.Anti-lipase and lipolytic activities of ursolic acid isolated from the roots of Actinidia arguta[J]. Arch Pharm Res, 2009, 32(7):983-987.
The aim of this study was to investigate the anti-obestic effects of ursolic acid isolated from the roots of Actinidia arguta , as well as the mechanism of action of this compound. This was conducted by testing whether ursolic acid inhibited the elevation of the rat plasma triacylglycerol levels after oral administration of a lipid emulsion containing corn oil in rats. Ursolic acid prevented the elevation of plasma triacylglycerol levels 2 h after oral administration of the lipid emulsion at a dose of 100 mg/kg. Furthermore, ursolic acid inhibited phosphodiesterase activity in vitro with an IC 50 of 51.21 M and enhanced lipolysis in rat fat cells. We suggest that the inhibitory effects of ursolic acid, isolated from the roots of A. arguta , on obesity, might be attributable to the inhibition of lipid absorption through the inhibition of pancreatic lipase and by enhancing lipolysis in fat cells.
JAYAPRAKASAMB, OLSON LK, SCHUTZKI RE, et al.Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas)[J]. J Agric Food Chem, 2006, 54(1):243-248.
Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.
CHUX, HEX, SHIZ, et al.Ursolic acid increases energy expenditure through enhancing free fatty acid uptake and β-oxidation via an UCP3/AMPK-dependent pathway in skeletal muscle[J]. Mol Nutr Food Res, 2015, 59(8):1491-1503.
Ursolic acid (UA) is a triterpenoid compound with multifold biological functions. Our previous studies have reported that UA protects against high‐fat diet‐induced obesity and improves insulin resistance (IR). However, the potential mechanisms are still undefined. Free fatty acid (FFA) metabolism in skeletal muscle plays a central role in obesity and IR. Therefore, in this study, we investigated the effect and the potential mechanisms of UA on skeletal muscle FFA metabolism.
JIAY, KIMS, KIMJ, et al.Ursolic acid improves lipid and glucose metabolism in high-fat-fed C57BL/6J mice by activating peroxisome proliferator-activated receptor alpha and hepatic autophagy[J]. Mol Nutr Food Res, 2015, 59(2):344-354.
Abstract SCOPE: This study investigated metabolic effects of ursolic acid (UA), a peroxisome proliferation-activated receptor (PPAR)-α activator, in vivo. METHODS AND RESULTS: High-fat diet (HFD)-fed C57BL/6J mice were orally administered UA (50 or 20002mg/kg body weight) for 8 wk. UA reduced liver and adipose tissue mass, adipocyte size, and plasma leptin concentrations, plasma triglyceride and low-density-lipoprotein cholesterol concentrations, while it elevated the high-density-lipoprotein cholesterol and adiponectin concentrations significantly compared with controls. UA induced the expression of PPARα and its responsive genes involved in fatty acid uptake and β-oxidation in the livers, whereas genes involved in lipogenesis, including sterol regulatory element-binding proteins-1c, were downregulated. UA administration improved glucose tolerance and insulin sensitivity significantly compared with the HFD-fed control livers. UA administration also activated hepatic autophagy as assessed by the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II and other key proteins in the autophagy pathway. CONCLUSION: Our findings suggest that UA ameliorates lipid and glucose metabolism in HFD-fed mice primarily by the activation of PPARα and induction of the hepatic autophagy pathway. Thus, intake of UA in the diet or in an isolated form may ameliorate lipid and glucose metabolism. 08 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
LIS, LIAOX, MENGF, et al.Therapeutic role of ursolic acid on ameliorating hepatic steatosis and improving metabolic disorders in high-fat diet-induced non-alcoholic fatty liver disease rats[J]. PLoS One, 2014, 9(1):e86724.
Background Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases around the world, and is closely associated with obesity, diabetes, and insulin resistance. Ursolic acid (UA), an ubiquitous triterpenoid with multifold biological roles, is distributed in various plants. This study was conducted to investigate the therapeutic effect and potential mechanisms of UA against hepatic steatosis in a high-fat diet (HFD)-induced obese non-alcoholic fatty liver disease (NAFLD) rat model. Methodology/Principal Findings Obese NAFLD model was established in Sprague-Dawley rats by 8-week HFD feeding. Therapeutic role of UA was evaluated using 0.125%, 0.25%, 0.5% UA-supplemented diet for another 6 weeks. The results from both morphologic and histological detections indicated that UA significantly reversed HFD-induced hepatic steatosis and liver injury. Besides, hepatic peroxisome proliferator-activated receptor (PPAR)-伪 was markedly up-regulated at both mRNA and protein levels by UA. Knocking down PPAR-伪 significantly inhibited the anti-steatosis role of UA in vitro . HFD-induced adverse changes in the key genes, which participated in hepatic lipid metabolism, were also alleviated by UA treatment. Furthermore, UA significantly ameliorated HFD-induced metabolic disorders, including insulin resistance, inflammation and oxidative stress. Conclusions/Significance These results demonstrated that UA effectively ameliorated HFD-induced hepatic steatosis through a PPAR-伪 involved pathway, via improving key enzymes in the controlling of lipids metabolism. The metabolic disorders were accordingly improved with the decrease of hepatic steatosis. Thereby, UA could be a promising candidate for the treatment of NAFLD.
SUNDARESANA, HARINIR, PUGALENDI KV.Ursolic acid and rosiglitazone combination alleviates metabolic syndrome in high fat diet fed C57BL/6J mice[J]. Gen Physiol Biophys, 2012, 31(3):323-333.
The aim of this study was to examine the combined effect of ursolic acid (UA) and rosiglitazone (RSG) on metabolic syndrome in C57BL/6J mice. Upon feeding high fat diet (HFD) C57BL/6J mice developed obesity, insulin resistance, dyslipidemia and hypertension. The male mice were randomly divided into six groups, and fed normal diet, normal diet plus UA and RSG, HFD alone, HFD plus UA, HFD plus RSG, and HFD plus UA and RSG, respectively. HFD fed mice showed increase in body weight, elevated plasma glucose and insulin. Activities of gluconeogenic enzymes such as glucose 6-phosphatase, fructose 1,6-bisphosphatase increased while the activity of glycolytic enzyme, glucokinase, decreased in the liver along with glycogen content. Total cholesterol, triglyceride, low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol and free fatty acid levels significantly increased in the plasma, whereas high-density lipoprotein cholesterol significantly decreased in high fat diet fed mice. In addition, both systolic and diastolic blood pressure was increased significantly. Combined treatment with UA and RSG improved the above parameters towards normality and pronounced more responses than UA or RSG lone treatment. The inclusion of UA in treatment with RSG may reduce the body weight gain, one of adverse side effect associated with the RSG-therapy.
JANG SM, KIM MJ, CHOI MS, et al.Inhibitory effects of ursolic acid on hepatic polyol pathway and glucose production in streptozotocin-induced diabetic mice[J]. Metabolism, 2010, 59(4):512-519.
The effects of ursolic acid on the polyol pathway and glucose homeostasis鈥搑elated metabolism were examined in the livers of streptozotocin (STZ)-induced diabetic mice fed a high-fat (37% calories from fat) diet for 4 weeks. Male mice were divided into nondiabetic, diabetic control, and diabetic鈥搖rsolic acid (0.05% wt/wt) groups. Diabetes was induced by the injection of STZ (200 mg/kg body weight, intraperitoneally). Although an ursolic acid supplement lowered the blood glucose level, it did not affect the plasma leptin and adiponectin levels. The present study shows that the blood glucose levels have a positive correlation with the hepatic sorbitol dehydrogenase activities ( r = 0.39, P < .05). Ursolic acid significantly inhibited sorbitol dehydrogenase activity as well as aldose reductase activity in the liver. The supplementation of ursolic acid significantly increased glucokinase activity, while decreasing glucose-6-phosphatase activity in the livers of STZ-induced diabetic mice. Ursolic acid significantly elevated the hepatic glycogen content compared with the diabetic control group. Supplementation with ursolic acid significantly lowered the plasma total cholesterol, free fatty acid, and triglyceride concentrations compared with the diabetic control group, whereas it normalized hepatic triglyceride concentration. A negative correlation was found between the hepatic triglyceride concentration and blood glucose levels ( r = 鈭0.50, P < .01) in regard to insulin-dependent diabetic mice. The hepatic fatty acid synthase activity was significantly lower in the ursolic acid group than in the diabetic control group, whereas hepatic fatty acid 尾 -oxidation and carnitine palmitoyltransferase activities were significantly higher. These results indicate that ursolic acid may be beneficial in preventing diabetic complications by improving the polyol pathway as well as the lipid metabolism and that it can function as a potential modulator of hepatic glucose production, which is partly mediated by up-regulating glucose utilization and glycogen storage and down-regulating glyconeogenesis in the liver.
WANG YL, WANG ZJ, SHEN HL, et al.Effects of artesunate and ursolic acid on hyperlipidemia and its complications in rabbit[J]. Eur J Pharm Sci, 2013, 50(3/4):366-371.
The hypolipidemic effect of artesunate is firstly reported. Its combination with ursolic acid might have the potential to further develop for the treatment of atherosclerosis.
Study of the Effectiveness of Additional Reductions in Cholesterol and Homocysteine (SEARCH) CollaborativeGroup, ARMITAGEJ, BOWMANL, et al.Intensive lowering of LDL cholesterol with 80 mg versus 20 mg simvastatin daily in 12 064 survivors of myocardial infarction: a double-blind randomised trial[J]. Lancet, 2010, 376(9753):1658-1669.
Lowering of LDL cholesterol reduces major vascular events, but whether more intensive therapy safely produces extra benefits is uncertain. We aimed to establish efficacy and safety of more intensive statin treatment in patients at high cardiovascular risk.We undertook a double-blind randomised trial in 12,064 men and women aged 18-80 years with a history of myocardial infarction. Participants were either currently on or had clear indication for statin therapy, and had a total cholesterol concentration of at least 3·5 mmol/L if already on a statin or 4·5 mmol/L if not. Randomisation to either 80 mg or 20 mg simvastatin daily was done centrally using a minimisation algorithm. Participants were assessed at 2, 4, 8, and 12 months after randomisation and then every 6 months until final follow-up. The primary endpoint was major vascular events, defined as coronary death, myocardial infarction, stroke, or arterial revascularisation. Analysis was by intention to treat. This study is registered, number ISRCTN74348595.6031 participants were allocated 80 mg simvastatin daily, and 6033 allocated 20 mg simvastatin daily. During a mean follow-up of 6·7 (SD 1·5) years, allocation to 80 mg simvastatin produced an average 0·35 (SE 0·01) mmol/L greater reduction in LDL cholesterol compared with allocation to 20 mg. Major vascular events occurred in 1477 (24·5%) participants allocated 80 mg simvastatin versus 1553 (25·7%) of those allocated 20 mg, corresponding to a 6% proportional reduction (risk ratio 0·94, 95% CI 0·88-1·01; p=0·10). There were no apparent differences in numbers of haemorrhagic strokes (24 [0·4%] vs 25 [0·4%]) or deaths attributed to vascular (565 [9·4%] vs 572 [9·5%]) or non-vascular (399 [6·6%] vs 398 [6·6%]) causes. Compared with two (0·03%) cases of myopathy in patients taking 20 mg simvastatin daily, there were 53 (0·9%) cases in the 80 mg group.The 6% (SE 3·5%) reduction in major vascular events with a further 0·35 mmol/L reduction in LDL cholesterol in our trial is consistent with previous trials. Myopathy was increased with 80 mg simvastatin daily, but intensive lowering of LDL cholesterol can be achieved safely with other regimens.Merck; The Clinical Trial Service Unit also receives funding from the UK Medical Research Council and the British Heart Foundation.
LIU HH, LI JJ.Aging and dyslipidemia: a review of potential mechanisms[J]. Ageing Res Rev, 2015, 19(1):43-52.
Abstract Elderly adults constitute a rapidly growing part of the global population, thus resulting in an increase in morbidity and mortality related to cardiovascular disease (CVD), which remains the major cause of death in elderly population, including men and women. Dyslipidemia is a well-established risk factor for CVD and is estimated to account for more than half of the worldwide cases of coronary artery disease (CAD). Many studies have shown a strong correlation between serum cholesterol levels and risk of developing CAD. In this paper, we review the changes of plasma lipids that occur in men and women during aging and the potential mechanisms of age-related disorders of lipoprotein metabolism covering humans and/or animals, in which changes of the liver sinusoidal endothelium, postprandial lipemia, insulin resistance induced by free fatty acid (FFA), growth hormone (GH), androgen (only for men) and expression and activity of peroxisome proliferator-activated receptor 伪 (PPAR伪) are mainly focused. Copyright 漏 2014. Published by Elsevier B.V.
KIM DK, BAEK JH, KANG CM, et al.Apoptotic activity of ursolic acid may correlate with the inhibition of initiation of DNA replication[J]. Int J Cancer, 2000, 87(5):629-636.
Abstract Top of page Abstract MATERIAL AND METHODS RESULTS DISCUSSION Acknowledgements REFERENCES Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti-tumor activity. In this study, we investigated the pro-apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration- and time-dependent manner. Furthermore, 30 M of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase-3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c -dependent caspase-3 activation. In addition, cell-cycle analysis revealed that UA-treated cells were arrested predominantly in the G 0 and G 1 phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21 WAF1 , a cell-cycle regulator, was increased by UA, indicating that p21 WAF1 might mediate UA-induced cell-cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA-induced cell-cycle arrest. Taken together, we suggest that UA-induced cell-cycle arrest may be mediated by inhibition of DNA replication and the increase of p21 WAF1 expression, which induces the release of cytochrome c and the activation of caspase-3, leading to apoptosis of HepG2 cells. Int. J. Cancer 87:629鈥636, 2000. 漏 2000 Wiley-Liss, Inc.
LIJ, GUO WJ, YANG QY.Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15[J]. World J Gastroenterol, 2002, 8(3):493-495.
姝 AIM:Ursolic acid(UA)and oleanolic acid(OA)aretriperpene acids having a similar chemical structure and aredistributed wildly in plants all over the world.In recentyears,it was found that they had marked anti-tumor effects.There is little literature currently available regarding theireffects on colon carcinoma calls.The present study wasdesigned to investigate their inhibitory effects on humancolon carcinoma call line HCT15.METHODS:HCT15 calls were cultured with different drugs.The treated calls were stained with hematoxylin-eosin andtheir morphologic changes observed under a lightmicroscope.The cytotoxicity of these drugs was evaluatedby tetrazolium dye assay.Cell cycle analysis was performedby flow cytometry(FCM).Data were expressed as meansSEM and Analysis of variance and Student' t-test forindividual comparisons.RESULTS:Twenty-four to 72 h after UA or OA 60 mol/Ltreatment,the numbers of dead calls and call fragmentswere increased and most calls were dead at the 72 nd hour.The cytotoxicity of UA was stronger than that of OA.Seventy-eight hours after 50 mol/L of UA or OA treatment,a number of calls were degenerated,but call fragments wererarely seen.The IC_(50)values for UA and OA were 30 and 60mol/L,respectively.Proliferation assay showed thatproliferation of UA and OA-treated calls was slightlyincreased at 24 h and significantly decreased at 48 h and 60h,whereas untreated control calls maintained anexponential growth curve.Cell cycle analysis by FCMshowed HCT15 calls treated with UA 30 and OA 60 for 36 h and72 h gradually accumulated in G_0/G_1 phase(both drugs P0.05 for 72 h),with a concomitant decrease of call populationsin S phase(both drugs P0.01 for 72 h)and no detectableapoptotic fraction.CONCLUSION:UA and OA have significant anti-tumor activity.The effect of UA is stronger than that of OA.The possiblemechanism of action is that both drugs have an inhibitoryeffect on tumor call proliferation through call-cycle arrest.
BAEK JH, LEE YS, KANG CM, et al.Intracellular Ca2+ release mediates ursolic acid-induced apoptosis in human leukemic HL-60 cells[J]. Int J Cancer, 1997, 73(5):725-728.
Abstract The effect of ursolic acid (UA) on tumor cell apoptosis was investigated using HL-60 human promyelocytic leukemia cells as a model cellular system. Treatment with UA resulted in a concentration-dependent decreased cell viability assessed by MTT assay. UA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which UA induced cell death was through apoptosis. The intracellular Ca2+ level was increased by treatment with UA. Intracellular Ca2+ inhibitors, such as intracellular Ca2+-release blockers (dantrolene, TMB-8 and ruthenium red) and an intracellular Ca2+ chelator (BAPTA/AM), significantly blocked the UA-induced increased intracellular Ca+ concentration. These inhibitors also blocked the effects of UA on cell viability and apoptosis. These results suggest that enhanced intracellular Ca2+ signals may be involved in UA-induced apoptosis in HL-60 cells.
HUANG MT, HO CT, WANG ZY, et al.Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid[J]. Cancer Res, 1994, 54(3):701-708.
Abstract A methanol extract of the leaves of the plant Rosmarinus officinalis L. (rosemary) was evaluated for its effects on tumor initiation and promotion in mouse skin. Application of rosemary to mouse skin inhibited the covalent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibited tumor initiation by B(a)P and 7,12-dimethylbenz[a]anthracene (DMBA). Topical application of 20 nmol B(a)P to the backs of mice once weekly for 10 weeks, followed 1 week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks, resulted in the formation of 7.1 tumors per mouse. In a parallel group of animals that were treated topically with 1.2 or 3.6 mg of rosemary 5 min prior to each application of B(a)P, the number of tumors per mouse was decreased by 54 or 64%, respectively. Application of rosemary to mouse skin also inhibited TPA-induced ornithine decarboxylase activity, TPA-induced inflammation, arachidonic acid-induced inflammation, TPA-induced hyperplasia, and TPA-induced tumor promotion. Mice initiated with 200 nmol DMBA and promoted with 5 nmol TPA twice weekly for 19 weeks developed an average of 17.2 skin tumors per mouse. Treatment of the DMBA-initiated mice with 0.4, 1.2, or 3.6 mg of rosemary together with 5 nmol TPA twice weekly for 19 weeks inhibited the number of TPA-induced skin tumors per mouse by 40, 68, or 99%, respectively. Topical application of carnosol or ursolic acid isolated from rosemary inhibited TPA-induced ear inflammation, ornithine decarboxylase activity, and tumor promotion. Topical application of 1, 3, or 10 mumol carnosol together with 5 nmol TPA twice weekly for 20 weeks to the backs of mice previously initiated with DMBA inhibited the number of skin tumors per mouse by 38, 63, or 78%, respectively. Topical application of 0.1, 0.3, 1, or 2 mumol ursolic acid together with 5 nmol TPA twice weekly for 20 weeks to DMBA-initiated mice inhibited the number of tumors per mouse by 45-61%.
HUBACEK JA, BOBKOVAD.Role of cholesterol 7alpha-hydroxylase (CYP7A1) in nutrigenetics and pharmacogenetics of cholesterol lowering[J]. Mol Diagn Ther, 2006, 10(2):93-100.
The relationship between dietary composition/cholesterol-lowering therapy and final plasma lipid levels is to some extent genetically determined. It is clear that these responses are under polygenic control, with multiple variants in many genes participating in the total effect (and with each gene contributing a relatively small effect). Using different experimental approaches, several candidate genes have been analyzed to date.Interesting and consistent results have been published recently regarding the A-204C promoter variant in the cholesterol 7alpha-hydroxylase (CYP7A1) gene. CYP7A1 is a rate-limiting enzyme in bile acid synthesis and therefore plays an important role in maintaining cholesterol homeostasis. CYP7A1-204CC homozygotes have the greatest decrease in total cholesterol level in response to dietary changes in different types of dietary intervention studies. In contrast, one study has reported that the effect of statins in lowering low-density lipoprotein (LDL)-cholesterol levels was slightly greater in -204AA homozygotes. The CYP7A1 A-204C variant accounts for a significant proportion of the genetic predisposition of the response of plasma cholesterol levels.
GILARDIF, MITRON, GODIOC, et al.The pharmacological exploitation of cholesterol 7alpha-hydroxylase, the key enzyme in bile acid synthesis: from binding resins to chromatin remodelling to reduce plasma cholesterol[J]. Pharmacol Ther, 2007, 116(3):449-472.
Mammals dispose of cholesterol mainly through 7alpha-hydroxylated bile acids, and the enzyme catalyzing the 7alpha-hydroxylation, cholesterol 7alpha-hydroxylase (CYP7A1), has a deep impact on cholesterol homeostasis. In this review, we present the study of regulation of CYP7A1 as a good exemplification of the extraordinary contribution of molecular biology to the advancement of our understanding of metabolic pathways that has taken place in the last 2 decades. Since the cloning of the gene from different species, experimental evidence has accumulated, indicating that the enzyme is mainly regulated at the transcriptional level and that bile acids are the most important physiological inhibitors of CYP7A1 transcription. Multiple mechanisms are involved in the control of CYP7A1 transcription and a variety of transcription factors and nuclear receptors participate in sophisticated regulatory networks. A higher order of transcriptional regulation, stemming from the so-called histone code, also applies to CYP7A1, and recent findings clearly indicate that chromatin remodelling events have profound effects on its expression. CYP7A1 also acts as a sensor of signals coming from the gut, thus representing another line of defence against the toxic effects of bile acids and a downstream target of agents acting at the intestinal level. From the pharmacological point of view, bile acid binding resins were the first primitive approach targeting the negative feed-back regulation of CYP7A1 to reduce plasma cholesterol. In recent years, new drugs have been designed based on recent discoveries of the regulatory network, thus confirming the position of CYP7A1 as a focus for innovative pharmacological intervention.
LIT, MATOZELM, BOEHMES, et al.Overexpression of cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis[J]. Hepatology, 2011, 53(3):996-1006.
UNLABELLED: We reported previously that mice overexpressing cytochrome P450 7a1 (Cyp7a1; Cyp7a1-tg mice) are protected against high fat diet-induced hypercholesterolemia, obesity, and insulin resistance. Here, we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had two-fold higher Cyp7a1 activity and bile acid pool than did wild-type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild-type to chenodeoxycholic acid (54%) in Cyp7a1-tg mice. Cyp7a1-tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than did wild-type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1-tg mice but intestine fractional cholesterol absorption in Cyp7a1-tg mice remained the same as wild-type mice despite the presence of increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (adenosine triphosphate-binding cassette G5/G8 [ABCG5/G8], scavenger receptor class B, member 1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1-tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue-specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. CONCLUSION: This study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis.
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