Analysis of the Heterogeneity of the Tumor Microenvironment and the Prognosis and Immunotherapy Response of Different Immune Subtypes in Hepatocellular Carcinoma.

Purpose: The current clinical classification of hepatocellular carcinoma (HCC) cannot well predict the patient's possible response to the treatment plan, nor can it predict the patient's prognosis. We use the gene expression patterns of patients with hepatocellular carcinoma to reveal the heterogeneity of hepatocellular carcinoma and analyze the differences in prognosis and immunotherapy response of different immune subtypes. Methods: Firstly, using the hepatocellular carcinoma expression profile data of TCGA, combined with the single sample gene set enrichment analysis (ssGSEA) algorithm, the immune enrichment of the patient's tumor microenvironment was analyzed. Subsequently, the spectral clustering algorithm was used to extract different classifications, and the cohort of hepatocellular carcinoma was divided into 3 subtypes, and the correlation between immune subtypes and clinical characteristics and survival prognosis was established. The patient's risk index is obtained through the prognostic prediction model, suggesting the correlation between the risk index and various types of immune cells. Results: We can divide the liver cancer cohort into three subtypes: stromal cell activated immune-enriched type (A-IS), general immune-enriched type (N-IS), and non-immune-enriched type (non-IS). The 3-year survival rate of TCGA's A-IS is higher than that of N-IS and non-IS, and the three components are significantly different (p = 0.017). The 3-year survival rates of ICGC's A-IS and N-IS groups were higher than those of the non-IS group. The analysis of the correlation between the risk index and immune cells showed that the patient's disease risk was significantly positively correlated with cancer-associated fibroblast (CAF) stimulated cell, activated stroma cell, and anti-PD-1 resistant cell. Conclusion: The tumor gene expression characteristics of patients with hepatocellular carcinoma can be used as a basis for clinical patient classification. Different immune subtypes are closely related to survival prognosis. Different immune cell states of patients may lead to different disease risk levels. All these provide important references for the clinical identification and prognosis prediction of hepatocellular carcinoma.

Elevated phthalates' exposure in children with constitutional delay of growth and puberty.

BACKGROUND: Phthalates have been proven to be antiandrogenic, which may interfere with the timing of puberty. Children with Constitutional Delay of Growth and Puberty (CDGP) typically display short stature and pubertal delay. This study investigated whether phthalate's exposure was associated with CDGP, and evaluated the potential mediator role of testosterone. METHODS: In this case-control study, a total of 167 boys, including 57 boys with CDGP (cases) and 110 controls were enrolled. We measured six major phthalate metabolites in urine samples using high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). The serum testosterone level was determined by radioimmunoassay. RESULTS: Children in the CDGP group were determined to have significantly elevated urinary phthalates concentration compared with control subjects (total phthalates median: case, 107.00 ng/ml; control, 62.22 ng/ml, p = 0.001). After adjustment for BMI and other confounding factors: mono-n-butyl phthalate (MBP), monoethyl phthalate (MEP) and total phthalate concentrations were significantly negatively associated with serum testosterone level (MBP: ß = -45.7, p = 0.017; MEP: ß = -31.6, p = 0.022; total phthalates: ß = -24.6, p = 0.011); MBP, MEP, mono (2-ethylhexyl) phthalate (MEHP) and total phthalates were significantly associated with CDGP (odds ratio: MBP: 8.30, p = 0.002; MEP: 5.43, p = 0.002; MEHP: 3.83, p = 0.017; total phthalates: 9.09, p = 0.001). Serum testosterone level acted as a mediator of the association between phthalates' exposure and CDGP (p = 0.002) (proportion mediated: 34.4%). CONCLUSIONS: In this case-control study, elevated phthalates' level was detected in children with CDGP in Shanghai, China and phthalate level was associated with CDGP, which appeared to be mediated by circulating testosterone level.

[Study on the mechanism of di-2-ethylhexyl phthalate and cypermethrin inducing gonadal dysgenesis in the prepubertal male rats].

OBJECTIVE: To investigate the mechanism of di-2-ethylhexyl phthalate (DEHP) and cypermethrin (CYP) inducing gonadal dysgenesis in prepubertal male rats. METHODS: A total of 40 healthy 3-week-old specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into four groups: control group (corn oil), DEHP group (500 mg/kg, dissolved in corn oil), CYP group (80 mg/kg, dissolved in corn oil), and combined exposure group (exposed to 500 mg/kg DEHP and 80 mg/kg CYP, dissolved in corn oil). Rats were treated by gavage administration once a day for 30 days. Twenty-four hours after the last exposure, the animals were sacrificed. The body weight and the wet weight of testis were determined, and the weight coefficient of testis was calculated. Radioimmunoassay was used to determine serum testosterone level. Ultrastructural-level histopathological changes of the testis were examined by transmission electron microscopy. The mRNA and protein expression of follicle stimulating hormone receptor (FSHR), androgen binding protein (ABP), inhibin beta-B (INHBB) and vimentin (VIM) were analyzed by real-time PCR and Western blot, respectively. Factorial design analysis of variance was used to compare differences between groups; interaction diagrams were used to determine the interaction between DEHP and CYP. RESULTS: Compared with those of the control group, the testis weights and testis coefficients of the DEHP, CYP, and combined exposure groups significantly decreased by 39.3-59.2%and 19.7-58.6%, respectively, and all exposure groups showed significant reductions in serum level of testosterone, ranging from 49.1% to 62.7% (P < 0.05 or P < 0.01). And all the exposure groups showed different levels of ultrastructural damages in the testes. Compared with that in the control group, the mRNA expression of FSHR, ABP, INHBB, and VIMin the DEHP group was down-regulated by 1.72, 2.64, 2.83 and 1.79 times, and their protein levels were significantly reduced by 65.2%, 53.7%, 70.1%, and 51.9% (P < 0.05 or P < 0.01). Significant decreases in mRNA expression of ABP (down 1.72 times) and INHBB (down 2.06 times) were observed in the CYP group, and their protein levels decreased by 38.3% and 49.7%, respectively (P < 0.05). The combined exposure to both DEHP and CYP resulted in big decreases in the mRNA levels of FSHR (down 1.62 times), ABP (down 2.00 times), INHBB (down 2.35 times), and VIM (down 1.54 times) and protein levels of FSHR (down 52.1%), INHBB (down 53.9%), and VIM (down 58.8%) (P < 0.05). Factorial design analysis of variance showed that the combination of two substances had an antagonistic effect on the expression of ABP and INHBB (P < 0.05). CONCLUSION: DEHP and CYP, alone or combined, can lead to gonadal dysgenesis in prepubertal male rats. Both of them can disrupt functional mRNA and protein expression in Sertoli cells to certain levels. The combination of DEHP and CYP shows antagonistic effects, and DEHP has a stronger reproductive toxicity than CYP.

[Adverse effect of environmental endocrine disruptors on gonadal development of prepubertal male rats and therapeutic effect of bushen tianjing recipe on it].

OBJECTIVE: To verify the antagonistic effect of Bushen Tianjing Recipe (BTR) on environmental endocrine disruptors (EEDs) induced gonadal dysgenesis (GD) Sprague-Dawley (SD) male rat model. METHODS: Totally 70 3-week-old male SD rats were randomly divided into seven groups, i.e., the control group (fed with corn oil), the model A group [di-2-ethylhexyl-phthalate (DEHP) 500 mg/kg], the CM A group (fed with DEHP 500 mg/kg + BTR 40 mL/kg), the exposed group B (fed with CYP 80 mg/kg), the CM B group (fed with CYP 80 mg/kg + BTR 40 mL/kg), the model C group [fed with DEHP 500 mg/kg + CYP 80 mL/kg], the CM C group (DEHP 500 mg/kg + CYP 80 mg/kg + BTR 40 mL/kg), respectively, 10 in each group. All were administered with corresponding medication by gastrogavage, once daily, for total 30 days. Rats were killed 24 h after the last administration, and their body weight and wet testis weight were weighed. The coefficient of testis was calculated. The serum testosterone (T) level was measured by chemiluminescent immunoassay. The histopathologic tissue was prepared. The ultrastructural changes of genital cells were observed by electron microscope. RESULTS: Compared with the control group, there was no statistical difference in the body weight increase among all groups (P > 0.05). The time of testicular descent and preputial separation were significantly delayed in each exposed group (P < 0.01). In the exposed group A and the exposed group C, the wet weight of the testes was reduced and serum T level decreased (P < 0.01). The coefficient of testis significantly decreased in the exposed group A (P < 0.01). Compared with corresponding model group, the time of testicular descent and preputial separation were significantly fore-laid in each corresponding CM group (P < 0.01). The weight of the testes, the coefficient of testis, and the serum T level increased in the CM A group (P < 0.01). The serum T level obviously increased in the CM B group (P < 0.05). CONCLUSIONS: The GD rat model was successfully duplicated by using DEHP. EEDs were proved to have significant anti-androgen activities. BTR was verified to have significant antagonistic to its anti-androgen effect.

[Single and combined toxic effects of di-2-ethylhexyl phthalate and cypermethrin on fertility and development in the the prepubertal male rats].

OBJECTIVE: To investigate the separate and combined toxic effects of di-2-ethylhexylphthalate (DEHP) and cypermethrin (CYP) on prepubertal male rats. METHODS: A total of 24 healthy 3-week-old SPF male Sprague-Dawley rats were randomly divided into four groups. They were control group (fed with corn oil), DEHP group (500 mg/kg, dissolved in corn oil), CYP group (80 mg/kg, dissolved in corn oil), combine group (DEHP 500 mg/kg + CYP 80 mg/kg, dissolved in corn oil), respectively. Each group consists of 6 rats. All the rats were exposed to DEHP or CYP by the means of gavage administration, once a day for 30 days. They were executed 24 hours after the last administration, and their weight, wet weight of the testis and testis coefficient was calculated. The histomorphological examination of the testis was conducted and the serum testosterone level was measured by Radioimmunoassay. The activities of LDH, ACP, ALP and SDH were assessed in testis homogenate. RESULTS: Compared with control group, the time of testicular descent and preputial separation was significantly delayed and the anogenital distance was significantly shorter in DEHP group, CYP group and combine group (P < 0.05 or 0.01). In DEHP group, the wet weight of testis, testis coefficient and the serum level of testosterone were significantly decreased (P < 0.05). The activity of ALP, ACP and LDH in testis homogenate was significantly increased, but that of SDH was significantly decreased (P < 0.01). Histologic structure and ultrastructure of testis showed obvious abnormalities in DEHP group, CYP group and combine group. CONCLUSIONS: DEHP and CYP had obviously separate and combined reproductive toxicity to prepubertal male rats. Both of them could cause the degeneration, necrosis and dysfunction of the seminiferous epithelium, sertoli cells and leydig cells and resulted in the abnormal development and function of the male reproductive system. Rats exposed to DEHP alone showed main toxic effect. There was no interactive influence on reproductive toxicity between DEHP and CYP in the combine group.

[The establishment of environmental endocrine disruptors induced precocious puberty Macaca fascicularis model and the intervention of yin nourishing fire purging Chinese materia medica].

OBJECTIVE: To establish the environmental endocrine disruptors (EEDs) induced precocious puberty macaca fascicularis model, thus verifying the antagonism of yin nourishing fire purging Chinese materia medica (YNFPCMM) against the estrogenic effects. METHODS: (1) The female prepubescent monkeys at different months old were fed with high dose and low dose EEDs. By observing EEDs' estrogenic effects, the optimal age, the optimal exposure dose, and the optimal exposure time were determined. (2) Fifteen female prepubescent monkeys were randomly divided into the exposure group, the treatment group, and the control group, 5 in each group. Those in the exposure group were fed with EEDs such as nonylphenol (4-NP) and bisphenol A (BPA). Those in the treatment group were fed with EEDs and YNFPCMM. Those in the control group were fed with dissolvent such as corn oil. The therapeutic course was 4 weeks. By the end of the treatment, the serum estrogen level was measured and the maturation index of the vaginal keratinocytes was calculated. The uterus was extracted to measure the wet weight, the endometrial thickness, the ring-shaped myometrium thickness, the endometrial epithelial cells, and the glandular epithelium height. RESULTS: (1) Twenty month was the optimal exposure age. The low dose EEDs was the best exposure dose, and 4 weeks was the optimal exposure time. (2) Compared with the control group, the serum estrogen level obviously increased, the maturation index of the vaginal keratinocytes significantly increased, the wet uterus weight, the endometrial thickness, the ring-shaped myometrium thickness, the endometrial epithelial cells, and the glandular epithelium height significantly increased in the exposure group (P<0.05). Compared with the exposure group, the aforesaid indices significantly decreased in the treatment group (P<0.05). CONCLUSIONS: The EEDs induced precocious puberty macaca fascicularis model was successfully established in this study. The EEDs were proved to have estrogenic-like activities. YNFPC-MM was proved to have significant antagonism like estrogenic activities.

Oral arginine improves linear growth of long bones and the neuroendocrine mechanism.

OBJECTIVE: To investigate the effect of oral administration of arginine on linear growth of long bones in male pubertal rats and the underlying mechanisms, focusing on expression of genes related to the hypothalamus-pituitary growth axis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. METHODS: Rats were randomly divided into control and intervention groups. In the intervention group, arginine was solved in water (0.045 g L-arginine was mixed with 1 mL water) and administered in rats (10 mL/kg) through gastric perfusion once per day, for totally 28 d. Rats in the control group received normal saline treatment. Bone histomorphometry analysis was used to measure growth plate width and mineral apposition rate of the tibia, as well as trabecular bone volume fraction, osteoblast surface and osteoclast surface of the femur. Serum growth hormone (GH) concentration was determined by radioimmunoassay. Real-time PCR was used to measure the expression of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclases (sGCα1 and sGCß1), growth hormone-releasing hormone (Ghrh) and somatostatin (SS) in hypothalamus, as well as Gh in pituitary. Western blot was used to detect the protein levels of nNOS, sGCα1 and sGCß1 in hypothalamus. RESULTS: After treatment with arginine, the growth plate width of tibia and osteoblast surface of femur were increased (P < 0.05), and serum GH concentration was elevated (P < 0.05). Besides, mRNA and protein levels of nNOS and sGCα1 (P < 0.05), as well as the expression of Gh mRNA (P < 0.01), were significantly up-regulated, while the expression of SS mRNA was down-regulated (P < 0.05). CONCLUSION: Oral administration of arginine could improve linear growth of long bones by regulating mRNA expression of SS and Gh and inducing GH secretion, possibly via nNOS-NO-sGC-cGMP signal transduction pathway.

[Dectective of serum di-n-butyl phthalate and di-2-ethylhexyl phthalate level in normal children].

OBJECTIVE: To study on the level of serum di-n-butyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP) in normal children by using gas chromatography. METHODS: Gas chromatography method was established. DBP, DEHP in normal children were detected, and their reference level was calculated, DBP and DEHP level in different age and genders were analysed. RESULTS: DBP could be detected in 110 serum samples from total 310 samples, and DEHP could be detected in 118 serum samples from the total 310 samples. The detected contrations of DBP range from non detectable to 0.98 ng/ml, P75 was 0.68 ng/ml, P95 was 0.75 ng/ml. There was no difference in the detection ratios between females and males, while baby group detection ratios was lower than other groups. The detected contrations of DEHP range from non detectable to 0.94 ng/ml, P75 was 0.67 ng/ml, P95 was 0.74 ng/ml. There was no difference in the detection ratios between females and males. While there was difference among age groups. CONCLUSION: The study domonstrated that DBP and DEHP concentrations in normal children are very low. The refrence level of DBP was suggested as lower than 0.75 ng/ml, DHEP was suggested as lower than 0.74 ng/ml.

[Effect of Chinese herbs for nourishing shen-yin and removing xiang-fire on estrogen receptor expression in reproductive organ of rats contaminated with environmental endocrine disruptor].

OBJECTIVE: To investigate the effect of Chinese herbs for nourishing Shen-yin and removing Xiang-fire (NYRF) on estrogen receptor (ER) expression in uterus and ovary of rats contaminated with nonylphenol (NP) or its bisphenol A (BPA) mixture, for exploring the action mechanism of NYRF in antagonizing the estrogen-mimetic activity of environmental endocrine disruptors (EEDs). METHODS: EEDs contaminated female SD rats, 3-week old, were divided into two groups, the treated group fed with NYRF and the control group with corn oil during the same period of contaminating for 15 days. The wet weight (WW) and organ coefficient (OC) of uterus in rats, as well as the ER protein and mRNA expressions in rat's uterus and ovary were detected and compared. RESULTS: As compared with normal range, WW and OC increased significantly in the contaminated rats of the control group, with significantly down-regulated ER protein expression in uterus, and expressions of ER alpha and ER beta gene and protein in ovary (P<0.05). While in the treated group, the above-mentioned abnormalities of various indicators were markedly reversed to a certain extent (P<0.05). CONCLUSION: EEDs show estrogenic-mimetic action on productive organs, which could be antagonized by NYRF, resulting in the down-regulated mRNA and protein expressions of ER in reproductive organs, so as to reduce the sensibility of reproductive organs to EEDs, which is probably one of the acting mechanisms of NYRF.

[Study on the levels of the bisphenol A, octylphenol, 4-nonylphenol in serum of precocious girls].

OBJECTIVE: To investigate the role of bisphenol A (BPA), octylphenol (OP), 4-nonylphenol(4-NP) causing precocious girls. METHODS: The blood samples were collected from 110 cases of precocious girls and 100 cases of normal girls. The concentrations of BPA, OP, 4-NP in serum samples were measured by using high performance liquid chromatography (HPLC), the volume of the uteruse and ovary, the content of estradiol (E2) were determined at the same time. The contents of BPA, OP, 4-NP in blood serum of precocious girls and the indices of the target organs were analyed by using correlation. RESULTS: BPA and, OP were detected in blood serum for 40.9% and 33.6% of precocious girls respectively, BPA and OP were detected for only 2% and 5% in normal girls respectively, 4-NP was detected for 24.54% of precocious girls and 3% of normal girls. The ratio of BPA, OP, 4-NP in blood serum of precocious girls was higher than normal girls respectively (P < 0.01), BPA in blood serum of precocious girls patients was positive correlation with the volume of the uterus (r = 0.557, P < 0.05), and positive correlation with volume of the ovary (r = 0.469, P < 0.01). OP in blood serum of precocious girls patients was positive correlation with the volume of uterus (r = 0.576, P < 0.05), positive correlation with volume of ovary (r = 0.676, P < 0.01). CONCLUSION: Precocious girls patients were contaminated more severely than normal girls, and BPA, OP could influence the volume of the uterus and ovary, so it had some relationship with cause of precocious girls.

[Estrogen-like activity of 4-nonylphenol and bisphenol in immature SD rats].

OBJECTIVE: To investigate the estrogen-like activity of 4-NP and BPA in the immature SD rats. METHODS: The vehicle, ethinyl estradiol (EE, 0.3 mg/kg, positive control), 4-NP (50 mg/kg, 100 mg/kg, 200 mg/kg), bisphenol (BPA, 200 mg/kg, 400 mg/kg, 600 mg/kg) were given orally (by gavage) q.d. to immature female Sprague-Dawley rats on the 21st, 22nd, 23rd postnatal days. The rats were sacrificed 24 hours after the last administration. Uterine wet weight, uterine/body weight ratio, height of the luminal epithelium, thickness of the myometrium and protein level of proliferating cell nuclear antigen (PCNA) of rat uterine were analysed by one-way ANOVA. RESULTS: In animal experiments, uterine wet weight and uterine/body weight ratio were significantly higher in the 4-NP 200 mg/kg and 100 mg/kg, BPA 600 mg/kg and 400 mg/kg (P < 0.05). Height of luminal epithelium, thickness of myometrium, as well as protein level of PCNA of rat uterine were significantly increased in the three groups of 4-NP, the three groups of BPA, and the mixture group (P < 0.05), and a dose-response relationship was observed. CONCLUSION: 4-NP and BPA had weak estrogen-like activities. They were the main environmental endocrine disruptors, and could influence the healthy of immature animals and children.

[Regulative effects of Chinese herbs for nourishing yin and removing fire on gene expressions of estrogen receptor alpha, insulin-like growth factor-1 receptor, epithelial growth factor receptor and protein synthesis in epiphyseal growth plate of female pubertal rats].

OBJECTIVE: To investigate the effect of Chinese herbs for nourishing yin and removing fire (NYRF) on gene expressions of estrogen receptor alpha (ER alpha), insulin-like growth factor-1 receptor (IGF-1R) and epithelial growth factor receptor (EGFR) in the epiphyseal growth plate of the female pubertal rats. METHODS: The rats were randomly divided into the control group and the intervened group. Immunohistochemistry and realtime-PCR methods were used to measure the gene expression of ER alpha, IGF-1R and EGFR and their protein synthesis in epiphyseal growth plate. RESULTS: After being intervened with NYRF, the gene expressions of ER alpha and IGF-1R were down-regulated and their protein synthesis markedly reduced, while those of EGFR were unchanged. CONCLUSION: NYRF can modulate the development and maturation of bone by regulating the expressions of ER alpha and IGF-1R in the epiphyseal growth plate.

[Study on the di-n-butyl phthalate and di-2-ethylhexyl phthalate level of girl serum related with precocious puberty in Shanghai].

OBJECTIVE: To determine the levels of the di-n-butyl phthalate and di-2-ethylhexyl phthalate in the serum of precocious girls in Shanghai, and study the development relationship between DBP, DEHP and precocious girls. METHODS: The concentrations of two phthalates (di-n-butyl phthalate, DBP, di-2-ethylhexyl phthalate, DEHP) in serum of 110 precocious girls and 100 normal children were measured by using gas chromatography, in the same time, the volumes of the uteruses and ovaries for precocious girls and normal children were measured by B-ultrasound and then did correlation analysis between the concentrations of DBP, DEHP and the volumes of uteruses and ovaries of precocious girls. RESULTS: DBP and DEHP were detected for 27.3% and 22.7% in precocious girls respectively, DBP and DEHP were detected for only 4% and 3% in normal children respectively. From the ratio of DBP and DEHP in serum of precocious girls were higher than normal children. Precocious girls uteruses and ovaries volume were higher than those of normal children respectively (P < 0.05, P < 0.05). DBP in serum of precocious girls had positive correlation with the volumes of the uteruses (r = 0.456, P < 0.05), and had positive correlation with volumes of the ovaries (r = 0.378, P < 0.01). DEHP in serum of precocious puberty had positive correlation with the volumes of uteruses (r = 0.382, P < 0.05), and had positive correlation with volumes of ovaries (r = 0.689, P < 0.01). CONCLUSION: Precocious girls could be contaminated more severely than normal girls, and DBP, DEHP could influence the volumes of the uteruses and ovaries.

[Determination of four kinds of environmental endocrine disruptors in blood serum of precocious puberty patients by high performance liquid chromatography].

OBJECTIVE: This study was directed to the development of a simple and highly sensitive method for determination of four kinds of environmental endocrine discrupters (EEDs) in the serum of precocious puberty patients using reversed-phase HPLC. METHODS: The serum samples were extracted by n-hexane. The operating conditions were such as C18 column (4.6 mm x 250 mm 5 microm), methanol: H2O: trifuorocuetic acid (1000 : 50 : 1) as mobile phase at a flow rate of 1 ml/min. Using ultraviolet detector, DEHP and MEHP was determined with wavelength at 228 nm, p, p'-DDE was determined with wavelength at 238 nm. Fluorescence detector with the excitation and emission wavelength at 275 nm and 312 nm respectively were used to determine 4-NP. RESULTS: There was a good linear relationship between the concentrations of the analytes in serum and their peak areas in the range of for DEHP, for MEHP, for p, p'-DDE and for 4-NP. The detection limit of the method was 1 ng/ml for each of the analytes. Recoveries of serum samples were 96.84% - 103.44% for DEHP, 87.69% - 104.92% for MEHP, 87.6% - 109.89% for p, p'-DDE, 98.57% - 105.1% for 4-NP. CONCLUSION: The method is suitable for the determination of the four kinds of EEDs in serum. It is easy to dispose the samples. The volume of samples is decreased and the effect of detection is increased. The method is quicker and easier and could be widely used to determine these compounds. It is helpful for the clinical study of precocious puberty.

[Study on the level of environmental endocrine disruptors in serum of precocious puberty patients].

OBJECTIVE: To investigate the role of environmental endocrine disruptors (EEDs) in causing the precocious puberty. METHODS: The blood samples were collected from 79 cases of precocious puberty patients and 42 cases of normal children. The concentrations of 4-nonylphenol (4-NP), 1, 1-dichloro-2, 2, bis (p-chlorophenyl) ethylene (p, p'-DDE) and di-2-ethylhexyl phthalate (DEHP) in blood serum samples were measured by using reversed-phase high performance liquid chromatography (HPLC). The volume of uterus and ovary, the bone density, and the content of estradiol (E(2)) in serum were determined at the same time. The contents of EEDs in blood serums of precocious puberty and the indices of the target organs were analyzed by using of correlation and regression. RESULTS: In normal control group, p, p'-DDE was detected in all the blood samples (14.93 - 40.39 ng/ml), but 4-NP and DEHP were detected in some samples (ND -6.77 ng/ml, ND -17.61 ng/ml). The levels of 4-NP, p, p'-DDE and DEHP in blood serum in precocious puberty group were notably increased than that in control group (P < 0.01). In precocious puberty group, there was a positive correlations between the 4-NP in volume of uterus and the volume of ovary and the density of bone (r = 0.394, 0.286, 0.237, P < 0.01); p, p'-DDE and volume of uterus also showed a the positive correlation (r = 0.306, P < 0.01). The influencing extent of 4-NP was 1.3 times to that of the p, p'-DDE. CONCLUSION: The normal children and the children with precocious puberty should be all contaminated by EEDs, and the later be exposured to more EEDs. There might exist a close relationship between EEDs and the precocious puberty, and EEDs should be an important factor in causing the disease. Different kinds of EEDs might have different influencing extents to the target organs.

[Effects of Chinese herbal medicine on modulating the course of puberty development in children with precocious puberty].

OBJECTIVE: To explore the therapeutic effects of Ziyin Xiehuo Recipe (ZYXHR) for nourishing yin and lowering fire and Yishen Tianjing Recipe (YSTJR) for nourishing kidney and replenishing essence on regulating the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis, and to reveal the mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development of children with precocious puberty. METHODS: The pubertal rats were fed with ZYXHR or YSTJR for 30 days, and the parameters of rats were monitored as the followings: The content of gonadotropin-releasing hormone (GnRH), the frequency and amplitude of GnRH impulse releasing, the releasing amounts of aminoacid neurotransmitters, and neuropeptide Y (NPY) and beta-endorphin (beta-END) in the gonadotrophic area of the hypothalamus were detected with neurobiological methods (push-pull perfusion, homogenate, incubation of brain slices, and immunohistochemical staining). The levels of gene and protein expressions of GnRH, growth hormone releasing hormone (GHRH) and somatostatin (SS) in hypothalamus, and follicular stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) in adenohypophysis as well as insulin-like growth factor I (IGF-I) in metaphysis were determined with real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The ZYXHR could reduce the activity of GnRH neurons in hypothalamus through inhibiting the release of central exciting aminoacid neurotransmitters, whereas promoting the release of central inhibiting aminoacid neurotransmitters, NPY and beta-END in gonadotrophic area of hypothalamus. The expression levels of GnRH, FSH and LH mRNAs were down-regulated while the expression level of SS mRNA in hypothalamus was up-regulated in the ZYXHR-treated group. The GH mRNA in hypophysis and the IGF-I mRNA in metaphysis were both down-regulated by ZYXHR. The YSTJR could promote the activity of GnRH neurons in hypothalamus through inhibiting the release of NPY in gonadotrophic area of hypothalamus, up-regulating the expression levels of GnRH, FSH, LH and GH mRNAs in hypophysis, and IGF-I mRNA in metaphysis, while down-regulating the expression level of SS mRNA in hypothalamus. CONCLUSION: The ZYXHR and YSTJR could both regulate the gonadotrophic and somatotrophic functions of hypothalamic-pituitary axis through modulating the neuroendocrine regulation and the gene expressions of GnRH and SS in hypothalamus, GH, FSH and LH in hypophysis, and IGF-I in metaphysis. These may be the chief mechanisms of ZYXHR and YSTJR in modulating the course of pubertal development and ameliorating the skeletal development in children with precocious puberty.

Evaluation of the true precocious puberty rats induced by neonatal administration of Danazol: therapeutic effects of nourishing "Yin"-Removing "Fire" Chinese herb mixture.

BACKGROUND: Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture, a traditional herb-based formulation, has been successfully used for the management of idiopathic true precocious puberty (IPP) for more than thirty years. Precocious puberty rat model by neonatal administration of Danazol was used to investigate the effects of the herb mixture on the advanced sexual development of the rats, and the expression of hypothalamic gonadotropin-releasing hormone (GnRH), which is the important regulator for the hypothalamus-pituitary-gonadal axis, particularly at puberty. METHODS: Female Sprague-Dawley rats were divided into five groups: intact normal (N), IPP model (M), vehicle with no IPP (V), IPP model exposed to herb mixture (HM) and IPP model exposed to saline (S). Rats at 5 days of age were given a single subcutaneous injection of 300 microgram of Danazol dissolved in 25 microliter vehicle of propylene glycol-ethanol (1:1, v/v), to establish the precocious puberty model. From the day 15, rats in HM and S groups were continuously fed with either Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture 2 ml or saline 2 ml, until 3 consecutive regular estrous cycles were established. The day of vaginal opening and the day of setup regular estrous cycle of the rats were observed. Blood concentration of estrogen was determined by radioimmunoassay. Immunohistochemistry and RT-PCR analysis were used to explore the expression of GnRH. RESULTS: The day of vaginal opening and first estrous showed significant advancement in M compared with N and V (p < 0.05, respectively). The blood estrogen level increased significantly in M compared with those in other groups (about 28 days of age, at the time of vaginal opening in M rats) (p < 0.05, respectively). GnRH cells in rostral medial septum (MS), Broca diagonal band nucleus (DBB) and the medial preoptic area (MPOA), were calculated. The number in M was less than those in N and V (p < 0.05, respectively). The number was significantly higher in HM than that in M (p < 0.05). The GnRH mRNA expression increased significantly in M compared with that in N and V (p < 0.05). CONCLUSION: The true precocious puberty model by neonatal administration of Danazol in female rats showed augmented expression of hypothalamic GnRH; the Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture down-regulated the increased GnRH expression, and significantly delayed the sexual development of the precocious puberty rat.

[Study on the regulative rule of reinforcing shen principle on sexual precocity and senescence at the molecular level].

The imbalance of Shen-yin and Shen-yang is a common pathological basis of sexual precocity and senescence. Senescence can be regarded as physiological Shen-deficiency, which is mainly the deficiency of Shen-Yang, while the essence of sexual precocity is hyperactivity of Fire due to Yin-deficiency, and deficiency of Shen-Yin is the predominance in the imbalance. The process of growth, development, maturing and aging led by Shen-qi, is mainly involving the hypothalamus-pituitary-growth hormone axis and sexual gland axis. Early initiation of these two axes appears in sexual premature children, manifesting syndrome of Fire hyperactivity due to Yin-deficiency, while in senescence, the decline of the axes appears ahead of time and manifests syndrome of Yang qi consumption. It has been shown in experimental and clinical researches that significantly good effects could be obtained by applying Chinese recipe for warmly reinforcing Shen-Yang to postpone senescence and recipe for nourishing Yin and removing Fire to inhibit sexual precocity. It also been proved by determining syndrome and speculating the damaged site according to the effects of drug that the pathologic damage of sexual precocity and senescence is sited on the hypothalamus or higher center.

Regulative actions of the Chinese drugs for tonifying the kidney on gene expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP.

It is found that the drugs for nourishing yin to reduce pathogenic fire can significantly down-regulate, and the drugs for tonifying the kidney to replenish essence can up-regulate mRNA expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP, indicating that the Chinese drugs for tonifying the kidney can regulate gene expression of the hypothalamic GnRH, pituitary FSH, LH, and osteoblastic BGP, which is possibly one of the main mechanisms of the Chinese drug for tonifying the kidney, regulating ephebic development process andimproving skeletal development in sexual precocity children.

[Pattern of abnormal skeletal development in girls with idiopathic precocious puberty and therapeutic effect of Chinese herbs for nourishing yin and purging fire].

OBJECTIVE: To explore the pattern of abnormal skeletal development in girls with idiopathic precocious puberty (IPP) and the therapeutic effect of Chinese herbs for nourishing yin and purging fire (CH). METHODS: Measurement of bone age, bone mineral content (BMC) and bone density (BD) were performed and levels of serum gla-protein (BGP), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP3) were determined in girls with IPP at various stages of pubertal development. Data were compared with those collected from matched girls, and the correlativity between various parameters and the extent of the disease were analyzed to explore the pattern. Thirty-eight girls out of them were treated with CH and the therapeutic effect was observed. RESULTS: The bone age of the IPP girls was ahead of time obviously (P < 0.05), levels of BMC, BD and serum BGP, IGF-1 were significantly higher than the levels in the healthy girls of matched age (P < 0.05). And the degree of these changes were markedly positive correlated with the severity of disease (P < 0.05). After being remitted with CH treatment, the advanced bone age was apparently alleviated, and above-mentioned abnormal criteria were lowered significantly (P < 0.05). CONCLUSION: The skeletal development of the girls with IPP was accelerated and the skeletal maturity were ahead of time, the more serious the condition of disease, the more obvious the acceleration and the advanced bone age. CH could decelerate skeletal development and delay the skeletal maturity of IPP patients.