This article investigated the effect and mechanism of salidroside(SAL) on the expression of adhesion molecules in oxidized low-density lipoprotein(oxLDL)-induced mouse aortic endothelial cell(MAEC). The oxLDL-induced endothelial cell injury model was constructed, and the safe concentration and action time of SAL were screened. The cells were divided into control group, oxLDL group, low and high concentration groups of SAL, and ferrostatin-1(Fer-1) group. The cell viability was detected by CCK-8 assay; lactate dehydrogenase(LDH) leakage was measured by colorimetry; the expression of intercellular adhesion molecule 1(ICAM-1) and recombinant vascular cell adhesion molecule 1(VCAM-1) were detected by immunofluorescence; Fe~(2+),glutathione(GSH),malondialdehyde(MDA),and 4-hydroxynonenal(4-HNE) levels were detected by kit method; reactive oxygen species(ROS) was detected by DCFH-DA probe; the levels of glutathione peroxidase 4(GPX4),silent mating type information regulation 2 homolog 1(SIRT1), and nuclear factor erythroid 2-related factor 2(Nrf2) were determined by using Western blot. The inhibitors of Nrf2 and SIRT1 were used, and endothelial cell were divided into control group, oxLDL group, SAL group, ML385 group(Nrf2 inhibitor), and EX527 group(SIRT1 inhibitor). The ultrastructure of mitochondria was observed by electron microscope; mitochondrial membrane potential(MMP) was detected by flowcytometry; the expressions of SIRT1,Nrf2,solute carrier family 7 member 11(SLC7A11),GPX4,ferroportin 1(FPN1),ferritin heavy chain 1(FTH1),ICAM-1, and VCAM-1 were detected by Western blot. The results showed that similar to Fer-1,low and high concentrations of SAL could improve cell viability, inhibit LDH release and the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cells(P<0.05 or P<0.01). It was related to increase in GSH level, decrease in Fe~(2+),ROS,MDA, and 4-HNE level, and up-regulation of SIRT1,Nrf2, and GPX4 expression to inhibit ferroptosis(P<0.05 or P<0.01). The intervention effect of high concentration SAL was the most significant. ML385 and EX527 could partially offset the protection of SAL on mitochondrial structure and MMP and reverse the ability of SAL to up-regulate the expression of SIRT1,Nrf2,SLC7A11,GPX4,FPN1, and FTH1 and down-regulate the expression of ICAM-1 and VCAM-1(P<0.05 or P<0.01).To sum up, SAL could reduce the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cell, which may relate to activation of SLC7A11/GPX4 antioxidant signaling pathway mediated by SITR1/Nrf2, up-regulation of FPN1 and FTH1 expression, and inhibition of ferroptosis.
Sirtuin 1/genetics* ; Animals ; Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; NF-E2-Related Factor 2/genetics* ; Mice ; Endothelial Cells/cytology* ; Glucosides/pharmacology* ; Phenols/pharmacology* ; Cell Adhesion Molecules/genetics* ; Reactive Oxygen Species/metabolism* ; Intercellular Adhesion Molecule-1/genetics* ; Vascular Cell Adhesion Molecule-1/genetics* ; Cell Survival/drug effects*

Objective To explore the role and mechanism of mammalian target of rapamycin (mTOR) in oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in vascular smooth muscle cells (VSMCs). Methods A model of oxLDL-induced VSMC ferroptosis was established. VSMCs were co-treated with either the mTOR inhibitor rapamycin or the autophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP), followed by detection of autophagy and ferroptosis-related indexes. Quantitative real-time PCR and Western blot were used respectively to analyze the expression of mTOR, glutathione peroxidase 4 (GPX4), sequestosome 1 (p62), and microtubule-associated protein 1 light chain 3 (LC3). Flow cytometry was employed to assess VSMC death. C11 BODIPY fluorescent staining was used to measure cellular lipid peroxidation levels. Colorimetric assays were performed to determine the contents of malondialdehyde (MDA), ferrous ion (Fe²⁺) and glutathione (GSH). Results oxLDL significantly upregulated mTOR expression in VSMCs, while increasing p62 expression and reducing LC3 expression, thereby suppressing VSMC autophagy. Compared with oxLDL treatment alone, rapamycin co-treatment reversed oxLDL-induced VSMC ferroptosis, as characterized by reduced VSMC death, increased GPX4 expression and GSH contents, along with decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Similarly, CCCP co-treatment activated autophagy characterized by reduced p62 expression and elevated LC3 expression, which subsequently alleviated oxLDL-induced ferroptosis, showing reduced VSMC death, increased GPX4 expressions and GSH contents, and decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Moreover, mTOR inhibition by rapamycin significantly reversed the oxLDL-induced upregulation of p62 and downregulation of LC3. Conclusion mTOR may promote oxLDL-induced VSMC ferroptosis by suppressing autophagy.
Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; TOR Serine-Threonine Kinases/physiology* ; Autophagy/drug effects* ; Muscle, Smooth, Vascular/metabolism* ; Animals ; Rats ; Myocytes, Smooth Muscle/cytology* ; Cells, Cultured ; Lipid Peroxidation/drug effects* ; Sequestosome-1 Protein/genetics* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Microtubule-Associated Proteins/genetics* ; Sirolimus/pharmacology*

Objective This study aims to investigate the effect of berberine (Ber) on foam cell formation induced by oxidized low-density lipoprotein (ox-LDL) in macrophages and to explore the mechanism's association with the ACE2-Ang(1-7)-Mas axis. Methods They were randomly divided into blank group, model group (RAW264.7 cells induced with 60 μg/mL ox-LDL), and berberine group (the model treated with berberine interventions at 2.5, 5, and 10 μmol/L concentrations). Lipid accumulation within the cells was assessed by Oil Red O staining, and the content of lipid droplets in each group was quantitatively analyzed by enzymatic method. The content of total cholesterol (TC) and free cholesterol (FC) in foam cells were detected by enzymatic method. The levels of oxidative stress factors (malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH)), inflammatory factors such as tumor necrosis factor α(TNF-α), and nitric oxide (NO) were measured using corresponding relevant reagent kits. The mRNA and protein expressions of ACE2 and Mas were evaluated through quantitative real-time PCR and Western blot analysis, respectively. The levels of AngII and Ang(1-7) were detected by ELISA. Results Compared with the model group, the berberine groups exhibited reduced lipid droplet accumulation and a dose-dependent decrease in intracellular lipid content. Berberine significantly lowered TC and FC levels in foam cells and reduced the CE/TC ratio. The levels of the oxidative factor MDA were significantly reduced, while the levels of the antioxidant factors SOD and GSH were markedly increased. Inflammatory factors TNF-α and NO were significantly decreased. The expression of the ACE2-Ang(1-7)-Mas signaling pathway was significantly activated, and the effect was more pronounced in the Ber group with high-concentration compared to the group with low-concentration, demonstrating a dose-dependent response. Conclusion Berberine can inhibit macrophage foam cell formation, potentially through upregulation of the ACE2-Ang(1-7)-Mas signaling pathway, thereby contributing to the alleviation of atherosclerosis.
Berberine/pharmacology* ; Foam Cells/cytology* ; Animals ; Signal Transduction/drug effects* ; Mice ; Angiotensin-Converting Enzyme 2 ; Angiotensin I/genetics* ; Peptidyl-Dipeptidase A/genetics* ; Peptide Fragments/genetics* ; Receptors, G-Protein-Coupled/genetics* ; RAW 264.7 Cells ; Proto-Oncogene Proteins/genetics* ; Proto-Oncogene Mas ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

The aim of this study was to investigate the underlying mechanism of chrysophanol(Chr) in reducing inflammation and foam cell formation induced by oxidized low-density lipoprotein(ox-LDL) and to investigate the targets and pathways related to effects of Chr on coronary atherosclerosis, providing a theoretical basis for the development of new clinical drugs. RAW264.7 macrophages were cultured in vitro, and after determining the appropriate concentrations of Chr and ox-LDL for treating RAW264.7 macrophages using a cell counting kit-8(CCK-8), the macrophages were treated with different concentrations of Chr(10, 15 μmol·L~(-1)) and ox-LDL(with or without 80 mg·mL~(-1)) for 24 h. RAW264.7 macrophages were divided into four groups: control group, model group(80 mg·mL~(-1) ox-LDL), treatment group(80 mg·mL~(-1) ox-LDL+10 μmol·L~(-1) Chr), and treatment group(80 mg·mL~(-1) ox-LDL+15 μmol·L~(-1) Chr). Lipid accumulation in each group was detected by oil red O staining. CD36 expression was analyzed by flow cytometry. Western blot was used to detect the expression of scavenger receptor class A1(SR-A1), scavenger receptor class B type Ⅰ(SR-B1), autophagy-related protein 5(Atg5), Beclin-1, autophagy adaptor protein p62(P62), the ratio of microtubule-associated protein light chain 3(LC3)Ⅱ to LC3Ⅰ(LC3Ⅱ/LC3Ⅰ), nuclear factor kappa B P65(NF-κB P65), inhibitor of κB kinase β(IKKβ), nuclear factor of κB inhibitor(IκB), high mobility group box protein 1(HMGB1), phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), and phosphorylated mammalian target of rapamycin(mTOR). Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression levels of ATP-binding cassette transporter A1(ABCA1), ATP-binding cassette transporter G1(ABCG1), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), HMGB1, inducible nitric oxide synthase(iNOS), arginase 1(Arg1), macrophage galactose-type lectin-1(Mgl-1), and NF-κB P65. Immunofluorescence analysis was performed to determine the localization of HMGB1 in RAW264.7 cells in each group. The autophagy inhibitor 3-methyladenine(3-MA) was added as a control for reverse validation, and the RAW264.7 macrophages were divided into four groups again: control group, model group(80 mg·mL~(-1) ox-LDL), treatment group(80 mg·mL~(-1) ox-LDL + 15 μmol·L~(-1) Chr), and inhibitor group(80 mg·mL~(-1) ox-LDL+15 μmol·L~(-1) Chr+3-MA). The results showed that Chr effectively reduced foam cell formation by regulating the expression levels of SR-A1, ABCA1, ABCG1, the LC3Ⅱ/LC3Ⅰ ratio, Atg5, Beclin-1, and p62, and inhibited the NF-κB/HMGB1-PI3K/Akt/mTOR signaling pathway. Moreover, the inhibitory effects of Chr on autophagy and the NF-κB/HMGB1-PI3K/Akt/mTOR pathway were reversed by the autophagy inhibitor 3-MA. In conclusion, Chr exhibits therapeutic potential for the treatment of atherosclerosis by inducing autophagy and modulating the NF-κB/HMGB1 and PI3K/Akt/mTOR pathways to inhibit the formation of macrophage inflammatory foam cells.
Animals ; Lipoproteins, LDL/metabolism* ; Mice ; TOR Serine-Threonine Kinases/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Macrophages/cytology* ; RAW 264.7 Cells ; Proto-Oncogene Proteins c-akt/genetics* ; Signal Transduction/drug effects* ; NF-kappa B/genetics* ; Anthraquinones/pharmacology* ; Foam Cells/cytology* ; HMGB1 Protein/genetics* ; Humans

The formation of macrophage-derived foam cells is a typical feature of atherosclerosis (AS). Reverse cholesterol efflux (RCT) is one of important factors for the formation of macrophage foam cells. In this study, macrophage form cells were induced by oxidized low density lipoprotein (ox-LDL) and then treated with different concentrations of ferulic acid, so as to observe the effect of ferulic acid on the intracellular lipid metabolism in the ox-LDL-induced macrophage foam cell formation, the cholesterol efflux and the mRNA expression and protein levels of ATP binding cassette transporter A1 (ABCA1) and ATP binding cassette transporter G1 (ABCG1) that mediate cholesterol efflux, and discuss the potential mechanism of ferulic acid in resisting AS. According to the findings, compared with the control group, the ox-LDL-treated group showed significant increase in intracellular lipid content, especially for the cholesterol content; whereas the intracellular lipid accumulation markedly decreased, after the treatment with ferulic acid. The data also demonstrated that the mRNA and protein expressions of ABCA1 and ABCG1 significantly increased after macrophage foam cells were treated with different concentrations of ferulic acid. In summary, ferulic acid may show the anti-atherosclerosis effect by increasing the surface ABCA1 and ABCG1 expressions of macrophage form cells and promoting cholesterol efflux.
ATP Binding Cassette Transporter 1 ; analysis ; genetics ; ATP Binding Cassette Transporter, Sub-Family G, Member 1 ; ATP-Binding Cassette Transporters ; analysis ; genetics ; Animals ; Cells, Cultured ; Cholesterol ; metabolism ; Coumaric Acids ; pharmacology ; Foam Cells ; drug effects ; metabolism ; Lipoproteins ; analysis ; genetics ; Mice

OBJECTIVETo observe the effect of Buyang Huanwu Decoction (BYHWD), a representative formula of qi benefiting blood activating method on aorta Rho associated coiled-coil forming protein serine/threonine kinase (Rhokinase, ROCK) and nuclear transcription factor kappa B (NF-κB) p65 mRNA expressions and levels of blood lipids in atherosclerosis (AS) model rats.

METHODSThe AS rat model was prepared by vitamin D3 and high fat diet. Totally 60 rats were randomly divided into 6 groups, i.e., the normal control group, the model group, the low dose BYHWD group (10 g/kg), the high dose BYHWD group (20 g/kg), the Simvastatin control group (0.6 mg/kg), and the BYHWD prevention group (10 g/kg), 10 in each group. After successful modeling all medication was intervened for 28 days. Expression levels oxidized low density lipoprotein (ox-LDL) were detected by ELISA. Levels of TG, TC, LDL-C, HDL-C were determined by enzyme method. Pathological changes of aortic tissue were observed under light microscope. mRNA expressions of Rho kinase and NF-κB p65 in aorta were detected by real time (RT) PCR.

RESULTSHigh fat diet and peritoneal injection of vitamin D3 could induce AS rat model. Typical atheromatous plaque formed in aorta of AS model rats. Compared with the normal control group, levels of TC, TG, LDL-C, and ox-LDL significantly increased in the model group, but the HDL-C level decreased (P < 0.01). Compared with the model group, levels of TC, TG, LDL-C, and ox-LDL all decreased, but HDL-C increased in low and high dose BYHWD groups, the Simvastatin control group, and the BYHWD prevention group (P < 0.05, P < 0.01). Compared with the low dose BYHWD group, above-mentioned indices were more obviously lowered in the high dose BYHWD group, the Simvastatin control group, and the BYHWD prevention group (P < 0.05). Compared with the normal control group, mRNA expression levels of Rho kinase and NF-κB p65 significantly increased in the model group (P < 0.01). Compared with the model group, mRNA expressions of Rho kinase and NF-κB p65 obviously decreased in low and high dose BYHWD groups, the Simvastatin control group, and the BYHWD prevention group (P < 0.01). Compared with the low dose BYHWD group, the two indicators were more obviously lowered in the high dose BYHWD group, the Simvastatin control group, and the BYHWD prevention group (P < 0.05). But there was no statistical difference in blood lipids levels, mRNA expression levels of Rho kinase or NF-κB p65 among the high dose BYHWD group, the Simvastatin control group, and the BYHWD prevention group (P >0. 05).

CONCLUSIONSBYHWD could down-regulate mRNA expression levels of Rho kinase and NF-κB p65, lower levels of blood lipids, and fight against AS. Suppressing Rho kinase pathway might be one of its mechanisms.

Animals ; Aorta ; Atherosclerosis ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression ; drug effects ; Lipids ; Lipoproteins, LDL ; NF-kappa B ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Simvastatin ; Transcription Factor RelA ; metabolism ; rho-Associated Kinases ; metabolism

To study the effects of alkaloids from Coptidis Rhizoma on low-density lipoprotein receptor (LDLR) mRNA expression and antihyperlipedemic levels. The LDLR mRNA expression were detected by real time fluorescence quantitative PCR, and the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in serum were measured at the first and last examination. The results show that, after the drug treatment, compared with the model group, each drug group showed a lipid-lowering effect. Especially, coptisine, palmatine, jatrorrhinze were significantly reduced TC, TG, LDL-c (P < 0.05, P < 0.01), and increased HDL-c (P < 0.01). In addition, they also increased mRNA expression of the LDLR in liver and HepG2 cells. The results showed that alkaloids from Coptidis Rhizoma can regulate lipid metabolism disorder, and coptisine have the best lipid-lowering effect.
Alkaloids ; administration & dosage ; Animals ; Cholesterol ; metabolism ; Cricetinae ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hyperlipidemias ; drug therapy ; genetics ; metabolism ; Hypoglycemic Agents ; administration & dosage ; Lipid Metabolism ; drug effects ; Lipids ; blood ; Lipoproteins, LDL ; metabolism ; Mesocricetus ; Receptors, Lipoprotein ; genetics ; metabolism ; Triglycerides ; metabolism

OBJECTIVETo investigate the effect of Antrodia cinnamomea on gene expression related to aortal endothelial injury of rats with hyperlipidemia.

METHODFifty SD rats were randomly divided into five groups: the normal control group (NG), the model group (MG), the antrodia cinnamomea groups of low, middle and high doses (AC-LG, AC-MG, AC-HG, 250, 500, 1 000 mg x kg(-1)). The rats were fed with high-fat diets to establish the hyperlipidemia model. After the drug administration for 10 weeks, their serum lipid, SOD, MDA and ox-LDL, LOX-1, P38 MAPK and NF-kappaB mRNA and protein expression were respectively determined, and the aortal endothelial injury was observed under electron microscope.

RESULTIn the model group, the contents of TC, TG and LDL-C significant increased (P < 0.01), whereas the content of HDL-C significant decreased (P < 0.01). Compared with the model group, both the AC-M group and the AC-H group showed reduction in endothelial injury and significant decrease in the content of TC, TG and LDL-C (P < 0.05 or P < 0.01). The content of HDL-C increased, but with no significant difference. SOD activity in serum remarkably increased (P < 0.05 or P < 0.01), MDA and ox-LDL levels dramatically decreased (P < 0.05 or P < 0.01).

CONCLUSIONA. cinnamomea can alleviate endothelial lipid injury by inhibiting the expressions of LOX-1, P38MAPK and NF-kappaB in aorta and better protect aortal endothelial cells from oxidative lipid injury.

Animals ; Antrodia ; chemistry ; Aorta ; drug effects ; metabolism ; ultrastructure ; Atherosclerosis ; blood ; genetics ; prevention & control ; Biological Products ; pharmacology ; Cholesterol ; blood ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Hyperlipidemias ; blood ; genetics ; prevention & control ; Lipoproteins, LDL ; blood ; Male ; Malondialdehyde ; blood ; Microscopy, Electron ; NF-kappa B ; blood ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class E ; blood ; genetics ; metabolism ; Superoxide Dismutase ; blood ; Triglycerides ; blood ; p38 Mitogen-Activated Protein Kinases ; blood ; genetics ; metabolism

We aimed to elucidate the effect of bilirubin on dyslipidemia and nephropathy in a diabetes mellitus (DM) type I animal model. Sprague-Dawley rats were separated into control, DM, and bilirubin-treated DM (Bil) groups. The Bil group was injected intraperitoneally with 60 mg/kg bilirubin 3 times per week and hepatoma cells were cultured with bilirubin at a concentration of 0.3 mg/dL. The Bil group showed lower serum creatinine levels 5 weeks after diabetes onset. Bilirubin treatment also decreased the amount of mesangial matrix, lowered the expression of renal collagen IV and transforming growth factor (TGF)-beta1, and reduced the level of apoptosis in the kidney, compared to the DM group. These changes were accompanied by decreased tissue levels of hydrogen superoxide and NADPH oxidase subunit proteins. Bilirubin decreased serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), free fatty acids, and triglycerides (TGs), as well as the TG content in the liver tissues. Bilirubin suppressed protein expression of LXRalpha, SREBP-1, SCD-1, and FAS, factors involved in TG synthesis that were elevated in the livers of DM rats and hepatoma cells under high-glucose conditions. In conclusion, bilirubin attenuates renal dysfunction and dyslipidemia in diabetes by suppressing LXRalpha and SREBP-1 expression and oxidative stress.
Animals ; Bilirubin/pharmacology/*therapeutic use ; Cell Line, Tumor ; Creatine/blood ; Diabetes Mellitus, Experimental/chemically induced/complications/*pathology ; Diabetic Nephropathies/*drug therapy/etiology ; Disease Models, Animal ; Kidney/pathology ; Lipoproteins, HDL/blood ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase/metabolism ; Orphan Nuclear Receptors/antagonists & inhibitors/genetics/metabolism ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Streptozocin/toxicity ; Triglycerides/analysis/*biosynthesis/blood

OBJECTIVETo investigate the effects of carbon disulfide (CS(2)) inhalation on the lipid levels of ApoE knockout gene mice and C57BL/6J mice.

METHODSFifty-one male ApoE gene knockout mice were randomly divided into four groups: CS(2)-exposed normal diet group, CS(2)-unexposed normal diet group, CS(2)-exposed high-fat diet group, and CS(2)-unexposed high-fat diet group. Fifty male C57BL/6J mice were divided into four groups in the same way. The exposed groups received 1000 mg/m3 CS(2) by static inhalation (5h/d, 5d/w) for four weeks. The weight of each mouse was determined and recorded once a week. On the 14th day of exposure, six mice in each group were randomly selected to measure serum total cholesterol (TC) levels. On the 28th day of exposure, the serum levels of TC and low-density lipoprotein (LDL) in the remaining mice were measured.

RESULTSThe mean weight gain of exposed groups was less than that of the unexposed groups. On the 14th and 28th days of experiment, the TC levels of the CS2-exposed high-fat diet group were significantly higher than those of the CS(2)-unexposed high-fat diet group among ApoE knockout gene mice (P < 0.01 for both). On the 14th day of experiment, the TC levels of the CS(2)-unexposed high-fat diet group were significantly higher than those of the CS(2)-unexposed normal-diet group among C57BL/6J mice group (P < 0.05). On the 28th day of experiment, the LDL levels of the CS(2)-exposed high-fat diet group were significantly higher than those of the CS(2)-unexposed high-fat diet group among ApoE knockout gene mice (P = 0.003).

CONCLUSIONCS(2) exposure, high-fat diet, and ApoE gene knockout can elevate blood lipids in mice, thus increasing the risk of atherosclerosis.

Administration, Inhalation ; Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; Body Weight ; Carbon Disulfide ; toxicity ; Diet, High-Fat ; adverse effects ; Gene Knockout Techniques ; Lipid Metabolism ; drug effects ; Lipids ; blood ; Lipoproteins, LDL ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout