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To protein extraction, and the amount of protein extracted was determined using a Bio-Rad protein assay kit. The other was cultured in a 24-well flat-bottomed [DTrp6]-LH-RH chemical information culture plate in serum-free Dulbecco’s modified Eagle’s medium supplemented with penicillin and streptomycin. After 12 hours, the supernatant was collected and the protein level measured. The amounts of TNF-a, IL6, and IL1-b proteins were measured using a Mouse ELISA Ready-SET-Go! kit according to the manufacturer’s protocol. Measurement of cytokine levels by enzyme-linked immunosorbent assay To determine the production and secretion of TNF-a protein in CDAA-treated mouse liver, a modified protocol that described in previous reports was used. In brief, a liver fragment was Mouse peritoneal macrophage experiments Mouse peritoneal macrophages were isolated from 8-week-old female C57BL/6J mice. Peritoneal cells were harvested by peritoneal lavage with 10 ml PBS. Cells were re-suspended and Nardilysin in NASH cultured in D-MEM supplemented with 10% FCS, 100 mg/ml of penicillin, 100 mg/ml of streptomycin, and 1.25 mg/ml of amphotericin B. 1.06106 peritoneal cells were seeded into a 48well dish, and incubated for 2 hours. Then, cells were washed in PBS, and re-cultured in the serum-free medium. To inhibit TNF-a activity, either control serum or 0.4 mg/ml of anti-TNF-a neutralizing polyclonal antibodies was administered into the culture medium. After 30 minutes later, 1 mg/ml of lipopolysaccharide were added. Medium and cells were collected 2 hours after the stimulation, and subjected to the analyses according to the methods described above. Statistical analyses Results are the mean 6 standard get SIS-3 deviation unless stated otherwise. Differences between treatments, groups, and strains were analyzed using the two-tailed Student’s t-test. Results Nrd12/2 mice did not develop steatohepatitis with CDAA diet The CDAA diet is deficient in choline only, but contains methionine, allowing observation of the sequential development of steatohepatitis and liver fibrotic changes in a longer experimental Nardilysin in NASH period in mice. The control CSAA diet also causes mild steatosis, but does not result in steatohepatitis and liver fibrotic changes in mice. To study the role of nardilysin during the development of steatohepatitis followed by liver fibrosis, Nrd1+/+ and Nrd12/2 mice were fed the CSAA or CDAA diets. Histology and oil red O staining showed that fat accumulation in the livers of both Nrd1+/+ and Nrd12/2 mice occurred during administration of the CDAA or CSAA diets and increased in a time-dependent manner, although fat accumulation in Nrd1+/+ mice was more prominent than that in Nrd12/2 mice. Size of fat deposition was greater in Nrd1+/+ mice than in Nrd12/2 mice in both diet groups at each time point, and triglyceride levels in the liver were significantly higher in Nrd1+/+ mice. There was no significant difference in the liver/body weight ratio between Nrd1+/+ and Nrd12/2 mice fed CSAA or CDAA diets. Thus, administration of CSAA or CDAA diets induced hepatic steatosis in mice to a varying degree. However, serum ALT levels were significantly increased in Nrd1+/+ mice upon administration of the CDAA diet, whereas they were not increased in Nrd12/2 mice fed the CDAA diet. Serum ALT level was elevated in neither Nrd1+/+ nor Nrd12/2 mice fed the CSAA diet. Consistent with these findings, qRT-PCR showed that mRNA expression of inflammatory cytokines, such as IL6 and IL1-b, was significantly incr.To protein extraction, and the amount of protein extracted was determined using a Bio-Rad protein assay kit. The other was cultured in a 24-well flat-bottomed culture plate in serum-free Dulbecco’s modified Eagle’s medium supplemented with penicillin and streptomycin. After 12 hours, the supernatant was collected and the protein level measured. The amounts of TNF-a, IL6, and IL1-b proteins were measured using a Mouse ELISA Ready-SET-Go! kit according to the manufacturer’s protocol. Measurement of cytokine levels by enzyme-linked immunosorbent assay To determine the production and secretion of TNF-a protein in CDAA-treated mouse liver, a modified protocol that described in previous reports was used. In brief, a liver fragment was Mouse peritoneal macrophage experiments Mouse peritoneal macrophages were isolated from 8-week-old female C57BL/6J mice. Peritoneal cells were harvested by peritoneal lavage with 10 ml PBS. Cells were re-suspended and Nardilysin in NASH cultured in D-MEM supplemented with 10% FCS, 100 mg/ml of penicillin, 100 mg/ml of streptomycin, and 1.25 mg/ml of amphotericin B. 1.06106 peritoneal cells were seeded into a 48well dish, and incubated for 2 hours. Then, cells were washed in PBS, and re-cultured in the serum-free medium. To inhibit TNF-a activity, either control serum or 0.4 mg/ml of anti-TNF-a neutralizing polyclonal antibodies was administered into the culture medium. After 30 minutes later, 1 mg/ml of lipopolysaccharide were added. Medium and cells were collected 2 hours after the stimulation, and subjected to the analyses according to the methods described above. Statistical analyses Results are the mean 6 standard deviation unless stated otherwise. Differences between treatments, groups, and strains were analyzed using the two-tailed Student’s t-test. Results Nrd12/2 mice did not develop steatohepatitis with CDAA diet The CDAA diet is deficient in choline only, but contains methionine, allowing observation of the sequential development of steatohepatitis and liver fibrotic changes in a longer experimental Nardilysin in NASH period in mice. The control CSAA diet also causes mild steatosis, but does not result in steatohepatitis and liver fibrotic changes in mice. To study the role of nardilysin during the development of steatohepatitis followed by liver fibrosis, Nrd1+/+ and Nrd12/2 mice were fed the CSAA or CDAA diets. Histology and oil red O staining showed that fat accumulation in the livers of both Nrd1+/+ and Nrd12/2 mice occurred during administration of the CDAA or CSAA diets and increased in a time-dependent manner, although fat accumulation in Nrd1+/+ mice was more prominent than that in Nrd12/2 mice. Size of fat deposition was greater in Nrd1+/+ mice than in Nrd12/2 mice in both diet groups at each time point, and triglyceride levels in the liver were significantly higher in Nrd1+/+ mice. There was no significant difference in the liver/body weight ratio between Nrd1+/+ and Nrd12/2 mice fed CSAA or CDAA diets. Thus, administration of CSAA or CDAA diets induced hepatic steatosis in mice to a varying degree. However, serum ALT levels were significantly increased in Nrd1+/+ mice upon administration of the CDAA diet, whereas they were not increased in Nrd12/2 mice fed the CDAA diet. Serum ALT level was elevated in neither Nrd1+/+ nor Nrd12/2 mice fed the CSAA diet. Consistent with these findings, qRT-PCR showed that mRNA expression of inflammatory cytokines, such as IL6 and IL1-b, was significantly incr.

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Author: haoyuan2014