Changes of free radicals and digestive enzymes in saliva in cases with deficiency inspleen-yin syndrome

Objective：To explore the nature of deficiency in spleen-yin syndrome,which could provide scientific theoretical support and practical guidance for clinical Traditional Chinese Medicine（TCM）syndrome differentiation based on biology,and had a strong clinical significance.Methods：Serum Cu and Zn were detected by atomic absorption spectrophotometer,serum vitamin E by high performance liquid chromatography,serum vitamin C by 2,4-Dinitrophenylhydrazine Colorimetry,total superoxide dismutase（SOD）and Cu and Zn- SOD by the xanthine oxidase method,and malondialdehyde（MDA）by the 2-thiobarbituric acid method（TBA）.Total antioxidant capacity was detected by a colorimetry kit.Amylase Activity was detected by an automatic biochemical analyzer.Lysozyme was detected by lysozyme detection plate,the diameter of bacteriolysis circle was measured and the corresponding content of lysozyme was obtained from a table of standard curve values.Results： No significant difference in total SOD and Cu,Zn-SOD was found between deficiency in spleen-yin group and normal group.However,such factors in deficiency in kidney-yin group were significantly lower than the other groups（P〈0.05）.The MDA content in both deficiency in spleen-yin group and deficiency in kidney-yin group were significantly higher than that of normal group（P〈0.05）,while the total antioxidant capacity was significantly lower than normal group（P〈0.05）.The vitamin E content in deficiency in kidney-yin group was significantly lower than that in the other two groups（P〈0.05）.No significant difference in the contents of vitamin C,Cu and Zn were observed in these groups.The Zn/Cu level in deficiency in kidney-yin group and the vitamin E level in deficiency in spleen-yin group decreased,but with no significant difference.Amylase activity in unit time in cases with deficiency in spleen-yin was lower than and had significant differences with that in normal cases,and higher than that in cases with deficiency in kidney-yin.The sectional velocity of saliva and the ratio of lysozyme in normal case group were significantly higher than other two groups,while deficiency in the spleen-yin group was significantly higher than the deficiency in kidney-yin group.Conclusion：All the results indicated that the objective pathological mechanism between the deficiency in spleen-yin and deficiency in kidney-yin was different.

Effects of Liuwei Dihuang Granule(六味地黄颗粒)on the Outcomes of In Vitro Fertilization Pre-Embryo Transfer in Infertility Women with Kidney-Yin Deficiency Syndrome and the Proteome Expressions in the Follicular Fluid

Objective： To observe the effects of Liuwei Dihuang Granule （六味地黄颗粒, LDG） for tonifying Kidney （Shen） on the outcomes of in v/tro fertilization pre-embryo transfer （IVF-ET） of infertility women with Kidneyyin deficiency syndrome and to explore its mechanism by detecting the proteome expression in the follicular fluid. Methods： Sixty-six infertility patients of Kidney-yin deficiency syndrome who would undergo IVF-ET, were randomly assigned to a treatment group and a control group according to a random number table, 33 cases in each group. Another 33 cases of non-Kidney-yin deficiency syndrome was taken as a syndrome-control group. Besides Westem routine therapy, LDG was given 3 menstrual cycles before IVF to the treatment group, and a placebo granule to the control and syndrome-control groups. The scores of Kidney-yin deficiency symptoms （sore waist and knees, dry vagina, dysphoria with feverish sensation in the chest, palms and soles, etc.） were assessed, the number of retrieved oocytes, rates of high quality oocytes and embryos, fertility rate and clinical pregnancy rate were recorded, and the follicular fluid was collected on the day when the ovum was picked up, the differential protein expression was detected using two-dimensional gel electrophoresis, and then, matrix assisted laser desorption ionization time-of flight mass spectrometry （MALDI-TOF-MS） was applied to identify the proteins. Results： The syndrome score in the treatment group decreased significantly from 16.09 ± 2.58 to 8.67 ± 2.13, while it changed insignificantly in the control group, with a significant difference in the lowering score between the two groups （P〈0.05）; the high quality rates of oocytes and embryos and clinical pregnancy rate were all superior in the treatment group to the control group （82.29% vs 78.08%, 76.76% vs 68.79%, 63.64% vs 36.36%, all P〈0.05）. The protein expression map from the follicular fluid showed that compared with the control group, 33 differential protein expressions were found in the syndrome-control group, among which 18 were down-regulated, and 15 up-regulated; in the treatment group 28 differential protein expressions were found, among which 15 were down-regulated, and 13 up-regulated. Through MALDI-TOF-MS, 14 proteins were identified （P〈0.05）. Conclusions： For the infertility patients undergoing IVF, LDG could alleviate clinical symptoms, improve rates of high quality oocytes and embryos, so as to raise clinical pregnancy rate. The mechanism may be through regulating proteome expression in the follicular fluid to improve the developmental microenvironment for oocytes which would lead to a successful embryo implantation.

Effects and related mechanisms of Huang's SiYu pills on Leydig cell function in kidney-yin deficiency rat models

The prescription“Huang's SiYu Pill”(HSYP)was founded by professor Huang Haibo,a famous traditional Chinese medicine in Inner Mongolia,based on long-term clinical experience.In this study we observed the effects of HYSP on sperm quality,hormone level and the expression level of reproductive related genes and proteins in testicular stromal cells of rats with kidney-yin deficiency to explore the therapeutic mechanism of HSYP in the treatment of infertility with kidney-yin deficiency,Liuwei Dihuang Pill group(LDP)and Tamoxifen Citrate Tablets combined with Pancreatic Kallidinogenase Enteric Coated Tablets were used to be the positive control drug.After 25 days of medicine administration,the animals were anesthetized and killed,the serum and testicular tissues were retained for further experiments.The ELISA results showed that HSYP had a better effect on increasing serum FSH level than the conventional Chinese medicine or western medicine,while there was no remarkable difference in the regulation of serum LH in each administration group.The results of RT-qPCR showed that the transcription levels of Cyp11a1,Hsd3b1,Lhcgr and Star genes in the medium and high dose groups of HSYP were significantly upregulated compared with the model group of kidney-yin deficiency,while the expression of Nr5a1 gene was not significantly affected.The results of HE and immunohistochemistry showed that HSYP significantly improved the development level of testicular stromal cells and promoted the expression levels of CYP11A1 and HSD11B1 in testicular stromal cells,compared with the model group of kidney-yin deficiency.Together,our results showed that Huang's SiYu Pills can significantly improve the development and function of Leydig cells in SD rats with kidney-yin deficiency model,which provided a good research basis for deep research on relevant pharmacological mechanisms of the medicine.