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Objective To establish a mouse model of kidney-yin and kidney?yang deficiency after oral administra?tion of hydrocortisone, and to explore the related evaluation factors. Methods The model was established by oral adminis?tration of hydrocortisone to induce kidney?yin and kidney?yang deficiency in mice. The survival and body weight of the mice were observed. The serum content of adrenal cortical hormone ( ACTH) , cortisol ( Cor. ) in the hypothalamic?pituitary?ad?renal (HPA) axis, and thyroid stimulating hormone (TSH), triiodothyronine (T3), thyrox (T4) in the hypothalamic?pi?tuitary?thyroid (HPT) axis, follicle?stimulating hormone (FSH), estradiol (E2), testosterone (T) in the hypothalamic?pituitary?gonadal ( HPG) axis were determined by radioimmunoassay. Results The body weight of kidney?yin and kidney?yang mice were decreased, the serum ACTH, Cor, TSH, T3, T4 contents were decreased, the serum FSH, E2, T contents were increased in the kidney?yang deficiency model mce ( P<0. 01 ) , and those parameters in the kidney?yin deficiency model mice were changed in opposite direction. Conclusions It is found that the hormone levels of ACTH, Cor, TSH, T3, T4, FSH, E2 and T in kidney deficiency mice are changed, and cortisol can be used as an important index to evaluate the model of kidney deficiency induced by glucocorticoid.
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Objective To observe the effects ofLiuwei Dihuang Decoction on cAMP and PDE3B in adipose tissues of rats with type 2 diabetes;To explore its mechanism.MethodsTotally 80 SD rats were randomly divided into two groups:control group and molding group. Rats in the molding group established the type 2 diabetic models by feeding high sugar and fat diet combined with intraperitoneal injection of Streptozotocin. The successful modeling rats were divided into model group, Rosiglitazone group, andLiuwei Dihuang Decoction group. Administration groups were given relevant medicine for gavage, while model group and blank group were given the same amount of normal saline for 30 days. FBG levels were detected, and cAMP and INS levels were detected by ELISA. The protein expressions of PDE3B in adipose tissues were determined by Western blot. The mRNA expressions of PDE3B in adipose tissues were detected by RT-PCR.Results Compared with the model group, the FBG and INS levels in type 2 diabetic rats were reduced in Rosiglitazone group andLiuwei DihuangDecoction group (P<0.05,P<0.01). InLiuwei Dihuang Decoction group, the protein and mRNA expressions of PDE3B in the adipose tissues were higher, and cAMP levels were lower than the model group (P<0.01).ConclusionLiuwei Dihuang Decoction can increase PDE3B expression, and decrease cAMP level, which may be one of the mechanisms for improving insulin resistance in type 2 diabetic rats.
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Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase(CBS) in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction (PCR) was employed to construct mutant plasmids pGEX4T-1-CBS(I278T) ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS (I278T) protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS(I278T) was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS (I278T ) protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine(AdoMet) with final concentration of 1 mmol/L showed no activation toward mutant CBS (I278T) protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS(I278T) had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .