Effect of Shuangyu Tiaozhi Decoction on NPC1L1 and ABCG8 in Intestine of Hyperlipemic Model Rats / 中国实验方剂学杂志

Objective:To observe the expression levels of niemann-pick C1-like 1 (NPC1L1) and adenosine triphosphate-binding cassette transporters G8 (ABCG8) in intestine of hyperlipidemic model rats, in order to investigate the therapeutic mechanism of Shuangyu Tiaozhi decoction on hyperlipidemia. Method:A total of 40 SD rats were selected, including 8 for normal control group. The remaining 32 rats were used to establish hyperlipemic model. After modeling, the rats were randomly divided into the model group (equivalent normal saline), the high and low-dose Shuangyu Tiaozhi groups (15.6, 7.8 g·kg-1), and the Simvastatin group (4 mg·kg-1), with 8 in each group. They were given drugs by gavage for 8 weeks. The levels of total cholesterol (TC), triglyceride (TG) in serum and total cholesterol (TTC), free cholesterol (FTC) in liver of rats in each group were determined by biochemical and enzymatic methods. The morphological changes of liver were observed by hematoxylin-eosin (HE) staining, and the levels of expressions of NPC1L1, ABCG8 and liver X receptor-α (LXR-α) in intestine were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot. The expression of ABCG8 protein was determined by immunohistochemistry. Result:After successful replication of the hyperlipidemia model, the blood lipid level was abnormally increased, and the liver steatosis became obvious in the model group compared with the normal control group. The expression levels of NPC1L1, LXR-α and ABCG8 increased significantly (PPα were significantly down-regulated, but ABCG8 was obviously up-regulated in a dose-dependent manner (PPPPPPConclusion:Shuangyu Tiaozhi decoction can reduce the blood lipid level of hyperlipemic rat model by reducing the absorption of cholesterol. Its mechanism may be correlated with the down-regulation of NPC1L1 expression and the up-regulation of ABCG8 expression.

Effects of berberine-evodiamine compatibility on expressions of intestinal ACAT2, ApoB48 and NPC1L1 in hypercholesterolemic rat / 中成药

AIM To investigate the effects of berberine-evodiamine compatibility on expressions of intestinal acyl-CoA:cholesterol acyltransferase 2 (ACAT2),apolipoprotein B48 (ApoB48),and Niemann-Pick C1 Like 1 (NPC1L1) proteins in hypercholesterolemic rats.METHODS Fifty SD rats were assigned to control and model groups.After establishing the hypercholesterolemic rat model by feeding high fat and high cholesterol food,forty SD rats were equally divided into model control group,atorvastatin group,berberine-evodiamine compatibility groups (89.2 mg/kg,178.4 mg/kg).After four weeks treatment,serum triglycerides (TG),total cholesterol (TC),low-density lipoprotein cholesterol (LDL-C),high density lipoprotein cholesterol (HDL-C) were detected.Then the contents of cholesterol and β-sitoesterol in serum were determined by GC.The expressions of ACAT2,ApoB48 and NPC1 L1 proteins in the small intestine were assayed with immunohistochemistry technology.RESULTS Serum TC,TG and liver TC were significantly decreased in 89.2 mg/kg and 178.4 mg/kg berberineevodiamine compatibility groups compared with those in the model control group (P ＜ 0.01).Serum LDL-C and liver TC were also significantly decreased in 89.2 mg/kg berberine-evodiamine compatibility group (P ＜ 0.05).GC analysis indicated that the amount of cholesterol level and β-sitoesterol in serum were decreased in 178.4 mg/kg berberine-evodiamine compatibility group (P ＜ 0.05) and 89.2 mg/kg berberine-evodiamine compatibility group (P ＜ 0.01).Immunohistochemistry analysis manifested that the average luminous density of ACAT2,ApoB48 and NPC1L1 proteins in two dosages of berberine-evodiamine compatibility group were descended markedly compared with those in the model control group (P ＜ 0.05 or P ＜ 0.01).CONCLUSION The mechanisms underlying the cholesterol metabolism activity of berberine-evodiamine compatibility are mediated most likely by down-regulating the expressions of intestinal ACAT2,ApoB48 and NPC1 L1 proteins in hypercholesterolemic rats.

Effects of berberine-evodiamine compatibility on expressions of intestinal ACAT2, ApoB48 and NPC1L1 in hypercholesterolemic rat / 中成药

AIM To investigate the effects of berberine-evodiamine compatibility on expressions of intestinal acyl-CoA:cholesterol acyltransferase 2 (ACAT2),apolipoprotein B48 (ApoB48),and Niemann-Pick C1 Like 1 (NPC1L1) proteins in hypercholesterolemic rats.METHODS Fifty SD rats were assigned to control and model groups.After establishing the hypercholesterolemic rat model by feeding high fat and high cholesterol food,forty SD rats were equally divided into model control group,atorvastatin group,berberine-evodiamine compatibility groups (89.2 mg/kg,178.4 mg/kg).After four weeks treatment,serum triglycerides (TG),total cholesterol (TC),low-density lipoprotein cholesterol (LDL-C),high density lipoprotein cholesterol (HDL-C) were detected.Then the contents of cholesterol and β-sitoesterol in serum were determined by GC.The expressions of ACAT2,ApoB48 and NPC1 L1 proteins in the small intestine were assayed with immunohistochemistry technology.RESULTS Serum TC,TG and liver TC were significantly decreased in 89.2 mg/kg and 178.4 mg/kg berberineevodiamine compatibility groups compared with those in the model control group (P ＜ 0.01).Serum LDL-C and liver TC were also significantly decreased in 89.2 mg/kg berberine-evodiamine compatibility group (P ＜ 0.05).GC analysis indicated that the amount of cholesterol level and β-sitoesterol in serum were decreased in 178.4 mg/kg berberine-evodiamine compatibility group (P ＜ 0.05) and 89.2 mg/kg berberine-evodiamine compatibility group (P ＜ 0.01).Immunohistochemistry analysis manifested that the average luminous density of ACAT2,ApoB48 and NPC1L1 proteins in two dosages of berberine-evodiamine compatibility group were descended markedly compared with those in the model control group (P ＜ 0.05 or P ＜ 0.01).CONCLUSION The mechanisms underlying the cholesterol metabolism activity of berberine-evodiamine compatibility are mediated most likely by down-regulating the expressions of intestinal ACAT2,ApoB48 and NPC1 L1 proteins in hypercholesterolemic rats.

Several proteins involved in absorption of cholesterol in small intestine / 军事医学

Several proteins are involved in the absorption of cholesterol in small intestine.Niemann-Pick C1 like 1 (NPC1L1) mainly mediates the absorption of cholesterol, and acyl-coenzyme A ( CoA)∶cholesterol acyltransferase 2 (ACAT2) catalyzes the free cholesterol absorpted by intestine into cholesterol ester,while unesterified free cholesterol is secreted into intestinal lumen by ATP-binding cassette(ABC) transporters G5/G8(ABCG5/ABCG8).Transcription factor liver X receptor( LXR) plays an important role in the process of intestinal cholesterol absorption.The research progress in NPC1L1,ABCG5/ABCG8,ACAT2 and LXR is reviewed in this article.

Effect of garlic extract on some serum biochemical parameters and expression of npc1l1, abca1, abcg5 and abcg8 genes in the intestine of hypercholesterolemic mice.

Some compounds in the garlic inhibit cholesterol synthesis, resulting in lowering of serum cholesterol and triglycerides and increase in HDL level. However, the mechanism of this specific effect is not fully understood. In the small intestine, ATP-binding cassette transporters G5, G8 and A1 (ABCG5, ABCG8 and ABCA1), as well as Niemann-Pick C1 like 1 (NPC1L1) protein have important roles in cholesterol metabolism. In this study, we evaluated the beneficial effect of aqueous extract of garlic on lipid profile and also expression of npc1l1, abca1, abcg5 and abcg8 genes in the intestine of N-Marry mice fed a high cholesterol diet as a possible mechanism of garlic effect. Twenty-four mice were randomly divided into three groups: Group 1: hypercholesterolmic (received chow + 2% cholesterol + 0.5% cholic acid); Group 2: garlic (received chow + 4% (w/w) garlic extract + 2% cholesterol + 0.5% cholic acid); and Group 3: received chow only. After one month, mice were anesthetized and blood was collected from their heart. The jejunum was removed, washed with PBS and entrocytes were scraped and used for the experiments. Serum lipids were measured enzymatically and expression of mRNA levels for the above-mentioned proteins was determined by semi-quantitative RT-PCR. Garlic extract significantly reduced serum lipids (p<0.05), compared with the hypercholesterolemic group. Expression of the intestinal npc1l1 was significantly decreased (p<0.01) in the garlic group, compared with the chow group, while abcg5 (p<0.01), abcg8 (p<0.01) and abca1 (p<0.05) expressions were significantly increased. In conclusion, this study reveals a possible mechanism for the beneficial effects of the garlic in lowering serum lipids by decreasing the intestinal lipid absorption and increasing excretion of cholesterol back into the intestinal lumen.

La respuesta terapéutica a ezetimiba en ratones C57BL/6 es mediada por cambios en la expresión de NPC1L1, ABCG5 y ABCG8 en el enterocito / Therapeutic response to ezetimibe in C57BL/6 mice is mediated by changes in NPC1L1, ABCG5 and ABCG8 expression in the enterocyte

Las proteínas NPC1L1, ABCG5 y ABCG8 participan en la absorción intestinal de colesterol. Ezetimiba inhibe este proceso bloqueando a NPC1L1, sin embargo, su efecto sobre ABCG5 y ABCG8 aún no está claro. Así, el objetivo del presente trabajo fue evaluar en ratones C57BL/6 con hipercolesterolemia inducida por dieta y tratados con ezetimiba, la expresión de NPC1L1, ABCG5 y ABCG8 mediante PCR en tiempo real y Western blot, en 3 grupos de animales: 1, dieta hipercolesterolémica D12336; 2, dieta D12336 más 5 mg/kg/día de ezetimiba; 3, dieta control. El nivel sérico de colesterol total fue significativamente diferente entre los grupos estudiados (control: 1,85 +/- 0,49 mmol/L; dieta D12336: 3,11 +/- 0,73 mmol/L; ezetimiba: 2,11 +/- 0,50 mmol/L, P = 0,001). La expresión génica de NPC1L1 aumentó 5,4 veces en el grupo que recibió la dieta D12336 (P = 0,003). Por otro lado, la expresión génica de ABCG5 y ABCG8 no fue diferente en el grupo con hipercolesterolemia (P = 0,239 y P = 0,201, respectivamente). Después del tratamiento con ezetimiba, la expresión génica de ABCG5 se incrementó 15,6 veces (P = 0.038). No hubo diferencias significativas en la expresión génica de NPC1L1 (P = 0,134) y ABCG8 (P = 0,067). En relación a la expresión proteica, la dieta D12336 incrementó los niveles de expresión de NPC1L1 (P = 0,022) y ABCG5 (P = 0,008); el tratamiento con ezetimiba incrementó los niveles de NPC1L1 (P = 0,048) y redujo los niveles de ABCG5 (P = 0,036) y ABCG8 (P = 0,016). En conclusión, nuestros resultados sugieren que tanto la dieta hipercolesterolémica como el tratamiento con ezetimiba, en un modelo experimental, afectan los niveles de expresión de NPC1L1, ABCG5 y ABCG8, sugiriendo que ABCG5 y ABCG8 están involucrados en la respuesta hipolipemiante a este fármaco. No obstante, el mecanismo mediante el cual se explica esta interacción requiere de un futuro estudio.

Proteins NPC1L1, ABCG5 and ABCG8 are involved in the intestinal absorption of cholesterol. Ezetimibe inhibits this process by blocking NPC1L1, however, its effect on ABCG5 and ABCG8 is not yet clear. Thus, the objective of this study was to evaluate in C57BL / 6 mice with diet-induced hypercholesterolemia treated with ezetimibe, the expression of NPC1L1, ABCG5 and ABCG8 by real time PCR and Western blot, in 3 groups of animals: 1, diet hypercholesterolemic D12336, 2, D12336 diet plus 5 mg/kg/ day of ezetimibe, 3, diet control. The serum level of total cholesterol was significantly different between groups (control: 1.85 +/- 0.49 mmol / L; diet D12336: 3.11 +/- 0.73 mmol / L; ezetimibe: 2.11 +/- 0.50 mmol / L, P = 0.001). NPC1L1 gene expression increased 5.4-fold in the group receiving the diet D12336 (P = 0.003). Furthermore, the gene expression of ABCG5 and ABCG8 was not different in the group with hypercholesterolemia (P = 0.239 and P = 0.201, respectively). After treatment with ezetimibe, ABCG5 gene expression was increased 15.6 times (P = 0.038). No significant differences in gene expression of NPC1L1 (P = 0.134) and ABCG8 (P = 0.067). Regarding protein expression, the D12336 diet increased the levels of expression of NPC1L1 (P = 0.022) and ABCG5 (P = 0.008), treatment with ezetimibe increased the levels of NPC1L1 (P = 0.048) and reduced levels of ABCG5 (P = 0.036) and ABCG8 (P = 0.016). In conclusion, our results suggest that both hypercholesterolemic diet as treatment with ezetimibe, in an experimental model, affect the expression levels of NPC1L1, ABCG5 and ABCG8, suggesting that ABCG5 and ABCG8 are involved in lipid-lowering response to this drug. However, the mechanism by which this interaction is explained requires further study.

Standardization of PCR-RFLP analysis of nsSNP rs1468384 of NPC1L1 gene.

Niemann-Pick C1-like 1 (NPC1L1) protein, a newly identified sterol influx transporter, located at the apical membrane of the enterocyte, which may actively facilitate the uptake of cholesterol by promoting the passage of sterols across the brush border membrane of the enterocyte. It effects intestinal cholesterol absorption and intracellular transport and as such is an integral part of complex process of cholesterol homeostasis. The study of population data for the distribution of these single nucleotide polymorphisms (SNP) of NPC1L1 has lead to the identification of six non-synonymous single nucleotide polymorphisms (nsSNP). The in vitro analysis using the software MuPro and StructureSNP shows that nsSNP M510I (rs1468384), which involves A→G base pair change leads to decrease in the stability of the protein. A reproducible and a cost-effective PCR-RFLP based assay was developed to screen for the SNP among population data. This SNP has been studied in Caucasian, Asian, and African American populations. Till date, no data is available on Indian population. The distribution of M510I NPC1L1 genotype was estimated in the North Western Indian Population as a test case. The allele distribution in Indian Population differs significantly from that of other populations. The methodology thus proved to be robust enough to bring out these differences.