Establishment Of Kidney-yang Deficiency Mouse Model By Inhibition Of Glucocorticoid Receptor Expression

Pattern of syndrome was the key point in basic theory and clinical applications of traditional Chinese medicine. Kidney-yang deficiency attracted great attention in recent yeares to make the syndrome more objective and reproducible.The previous studies showed that kidney-yang deficiency patients suffered from hypothalamic-pituitary-adrenocortical system(HPA) and three target glands(adrenal body, thyroid and sex gland) functional disorder. The medicine invigorating kidney-yang could elevate hypothalamus corticotropin releasing hormon level and improve the immunosuppressive action of glucocorticoid. The functional disorder of HPA and three target glands were considered as the base of kidney-yang deficiency. HPA and three target glands were a complex system ,so simple index need to be found for the benefit of clinical diagnosis and research. The research demonstrated that glucocorticoid receptor(GR) was an important hormone receptor in HPA and glucocorticoid which should be combined with GR to generate biological effect. Based on these findings above, we proposed following hypothesis: (1)GR down-regulation is one reason for kidney-yang deficiency; (2)GR could be one index of kidney-yang deficiency;(3) down-regulation of GR could be a method to construct kidney-yang deficiency model.There were some GR down-regulation methods: such as gene knockout, RNAi, AS-ODN and so on. Knockout of GR gene in mouse could make GR expression nearly disappear in all organs and tissues which would lead to the death of mouse before or after birth, so GR knockout could not imitate raw state of kidney-yang deficiency. RNAi could knockdown GR gene,but it were usually used as gene knockdown method in cells or animal gene knockdown just in short term. GR knockdown in long term can't achieve by RNAi recently. AS-ODN is one method of gene knockdown which could downregulate specific effective. But it`s half-period is short .The sustained release technique combined with AS-ODN made long-term gene knockdown in animal possible. It remain unclear that if delayed release technique combined with AS-ODN could knockdown GR expression in mouse .Objective: The aims of this study were:(1) AS-ODN was embeded in degradable polylactic acid microsphere to make prepare for the construction of GR knockdown mouse.(2) Several doses of microsphere embedded with AS-ODN were hypodermic injected to mouse to find the best GR knockdown dose.(3) Observe the relationship between GR downregulation and kidney-yang deficiency.Methords:(1) Design AS-ODN sequence base on the GR gene sequence and prepare degradable polylactic acid microsphere by lyopyilization.Observe the pharmacokinetics character of polylactic acid microsphere to make sure if AS-ODN could release slowly.(2) Different doses of polylactic acid microsphere with AS-ODN were hypodermic injected to C57 mouse to construct GR knockdown animal model . RT-PCR was used to distinguish a proper microsphere dose which can knockdown GR expression effectively.The proper dose would be used in the following experiment to construct GR knockdown animal model.(3) Construct GR knockdown mouse model and invigorate kidney-yang medicine were used to the mouse to see if it would upregulation GR expression. AS-ODN-loaded biodegradable Polylactic acid microspheres were prepared by double emulsion - solvent evaporation method.. The electron microscope observation showed that diameter of microspheres were between 10 to 20μm,and they take on round. Shape. Druge loading of the microspheres were between 6.0～11.5μg/mg.Downregulation of GR expression mouse model was constructed by hypodermic injection of AS-ODN-loaded biodegradable Polylactic acid microspheres.GRmRNA expression was decreased in liver,brain and kidney tissues.GRmRNA downregulation of 0.1 mg dose group mouse was between 23%~26% in the liver,brain and the kidney tissues;GRmRNA downregulation of 0.2 mg dose group mouse was between 36%~43%; GRmRNA downregulation of 0.4 mg dose group mouse was between 26%~41%; GRmRNA downregulation of 0.8 mg dose group mouse was between 32%~40%。GRmRNA expression were downregulated in different dose of AS-ODN group compared to the blank control group.The growth and development of mouse were inhibited due to hypodermic injection of AS-ODN-loaded microspheres.These mouse have shown kidney-yang deficiency pattern in their gross appearance such as pile,piloerection and so on .GRmRNA and GR protein were detected one week after hypodermic injection of AS-ODN-loaded microspheres and sense oligonucleotide-loaded microspheres respectively which indicated that AS-ODN group have less expression in both GRmRNA and GR protein than the sense oligonucleotid Group.GRmRNA in invigorating kidney-yang group downregulated placebo group 37%～41% compared to the sense oligonucleotide group. GRmRNA in placebo group downregulated 39%～41% compared to the sense oligonucleotide Group.GRmRNA in placebo group and invigorating kidney-yang group downregulated 34%～41% and 10%～25% respectively after placebo treatment and invigorating kidney-yang treatment for a month.GR protein expression showed the same tendency. One week after hypodermic injection of AS-ODN-loaded microsphere ,GR protein expression downregulated 52%～57% in placebo group, and GR protein expression downregulated 54%～61% in invigorating kidney-yang group. GR protein expression in placebo group and invigorating kidney-yang group downregulated 52%～61% and 16%～32% respectively after placebo treatment and invigorating kidney-yang treatment for one month.Weight growth velocity in invigorating kidney-yang group was faster than that of placebo group after invigorating kidney-yang treatment and placebo treatmen for a mouth .Conclusions:(1) Degradable polylactic acid microsphere with AS-ODN could knockdown GR expression in mouse by hypodermic injection.(2) GR knockdown C57 mouse could be a kidney-yang deficiency animal model.