| Objective:By observing the effect of Yougui Wan and its components on the energy metabolism of liver mitochondria in rats with kidney yang deficiency established by hydrocortisone method,the mechanism of"seeking Yang in Yin"of Yougui Wan was discussed,and the mechanism of energy metabolism in kidney yang deficiency was interpreted.The biology of"seeking Yang in Yin"method was revealed from the perspective of energy metabolism At the same time,it also provides experimental basis for the clinical application of wenbushenyang traditional Chinese medicine.Method:56 SD rats(male,200±20g)were fed adaptively for one week to prepare rats with kidney yang deficiency syndrome.According to the principle of random grouping,8 rats were served as blank control group,and the remaining 48 rats were divided into model group,Yougui Pill group,Ziyin group and Buyang group,with 12 rats in each group.After successful modeling,Yougui Pill group,Ziyin group and Buyang group were given 10.26,6.21,4.05g/kg by gavage respectively,once a day for 28 days,The volume of gavage was 7.5 ml/kg;the blank control group and model group were given the same volume of normal saline.1.On the 15th day of modeling,the body mass and Anal temperature of rats in the blank control group and model group were measured;the serum c AMP content and c GMP content of rats in the blank control group and model group were measured by ELISA,and the camp/c GMP ratio was calculated for model identification.2.After the last administration,the body weight and Anal temperature of rats in each group were measured to observe the weight growth and Anal temperature change of rats in each group.3.After fasting for 12 hours after gavage,blood was collected from inferior vena cava under anesthesia.Serum camp and c GMP were measured by ELISA,and the ratio of c AMP/c GMP was calculated.The structure of liver mitochondria was observed by electron microscope,and the crude mitochondria were prepared by liver homogenate.The activities of SDH,Na+-K+-ATPase,CCO,LDH and ATP content of liver mitochondria were measured by enzyme labeling method.Result:1.Changes of Anal temperature,body mass and c AMP/c GMP ratio in each group after interventionCompared with the blank control group,the anal temperature,weight and c AMP/c GMP ratio of the model group decreased significantly(P<0.05 or P<0.01).Compared with the model group,the rats in Yougui Wan group had higher Anal temperature,weight and c AMP/c GMP ratio(P<0.01);the rats in Buyang group had lower Anal temperature(P>0.05),higher body mass(P<0.05),higher c AMP/c GMP ratio(P<0.05);the rats in Ziyin group had no significant changes in Anal temperature,body mass and c AMP/c GMP ratio(P>0.05).Compared with the blank control group,the anal temperature of Yougui Wan group recovered,the body weight was similar to the normal level,the c AMP/c GMP ratio did not change significantly(P>0.05),the body weight of Buyang group was lower,and the c AMP/c GMP ratio decreased(P<0.05 or P<0.01).2.Changes of SDH activity in liver mitochondria of rats after intervention.Compared with the blank control group,the SDH activity in the model group decreased significantly(P<0.01).Compared with the model group,the activity of SDH in Ziyin group had little change(P>0.05),and that in Buyang group and Yougui Wan group increased(P<0.05 or P<0.01).Compared with the blank control group,the SDH activity of Buyang group and Yougui Wan group was slightly lower(P>0.05).Compared with the Buyang group,the SDH activity of Yougui Wan group was slightly higher,but the difference was not statistically significant(P>0.05).3.Changes of Na~+-K~+-ATPase activity in liver mitochondria of rats after interventionThe activity of Na~+-K~+-ATPase in the model group was significantly lower than that in the blank control group(P<0.01).Compared with the model group,Na~+-K~+-ATPase activity in Ziyin group,Buyang group and Yougui Pill group increased significantly(P<0.05 or P<0.01).Compared with the blank control group,Na~+-K~+-ATPase activity in Yougui Wan group returned to the normal level(P>0.05);Na~+-K~+-ATPase activity in Ziyin group and Buyang group was still at a low level(P<0.05 or P<0.01).4.Changes of CCO activity in liver mitochondria of rats after interventionCompared with the control group,CCO activity in the model group decreased significantly(P<0.01).Compared with the model group,the CCO activity of Ziyin group and Buyang group was slightly higher,but the difference was not statistically significant(P>0.05).After the intervention of Yougui Pill,the CCO activity was significantly higher than that of the model group(P<0.05);compared with the blank control group,the CCO activity was slightly lower,but the difference was not statistically significant(P>0.05).5.Changes of LDH activity in liver mitochondria of rats after interventionCompared with the blank control group,LDH activity in the model group decreased significantly(P<0.01).Compared with the model group,the LDH activity of Ziyin group,Buyang group and Yougui Wan group increased(P<0.05 or P<0.01).Compared with the blank control group,the activity of LDH in Buyang group and Yougui Wan group basically recovered to the normal level(P>0.05);the activity of LDH in Ziyin group was still at a low level(P<0.05).Compared with the Buyang group,the LDH activity of Yougui Pill was slightly higher,but the difference was not statistically significant(P>0.05).6.Changes of ATP content in liver mitochondria of rats after interventionCompared with the blank control group,ATP content in the model group was significantly lower(P<0.01).Compared with the model group,ATP content in Ziyin group,Buyang group and Yougui Pill group increased(P<0.05 or P<0.01).Compared with the blank control group,the content of ATP in Ziyin group was still lower(P<0.05);the content of ATP in Buyang group and Yougui Wan group basically returned to the normal level(P>0.05).Compared with Buyang group,the content of ATP in Yougui Pill was slightly higher,but the difference was not statistically significant(P>0.05).7.The changes of transmission electron microscope of liver tissue after interventionCompared with the blank control group,the mitochondrial structure of the model group was abnormal,some of them were vacuolated,even disappeared.Compared with the model group,the edge of mitochondrial membrane in Buyang group and Ziyin group recovered,the number of ridges increased,the arrangement was loose,but compared with the control group,the number of ridges decreased;the structure of mitochondria in Yougui Wan group basically recovered,the number of ridges increased.Conclusion:1.The ratio of c AMP/c GMP in serum of rats with kidney yang deficiency syndrome was significantly reduced,the activities of SDH,Na~+-K~+-ATPase,CCO,LDH and ATP content in liver mitochondria were significantly decreased,The ultrastructural damage of liver mitochondria is serious,and there was mitochondrial energy metabolism disorder,and a series of related symptoms such as hypofunction and slow metabolism appeared.2.After being treated with Yougui Wan,the ratio of c AMP/c GMP in serum of rats with kidney yang deficiency syndrome increased significantly;the activities of SDH,Na~+-K~+-ATPase,CCO and LDH in liver mitochondria increased significantly;the content of ATP in liver mitochondria recovered significantly;the pathological changes of liver mitochondria ultrastructure improved significantly;the disorder of energy metabolism and related physical signs recovered.This may be the mechanism of Yougui Wan in the treatment of kidney yang deficiency,and it is also one of the modern biological essence of the method of"seeking Yang from Yin",which also provides a scientific experimental basis for the application of wenbushenyang traditional Chinese medicine. |
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