[Xiongcan Yishen Formula up-regulates the clock gene expressions in the testis tissue of the rats with late-onset hypogonadism].

OBJECTIVE: To investigate the effect of Xiongcan Yishen Formula (XYF) on the expressions of the clock genes in the testis tissue of the rats with late-onset hypogonadism (LOH). METHODS: Forty-eight 8-week-old male SD rats were randomly divided into 6 groups, normal control, model control, testosterone propionate (TP), and low-, medium- and high-dose XYF. The LOH model was made in the later 5 groups of rats by intraperitoneal injection of D-galactose at 480 mg/kg/d for 56 successive days, while the normal controls were injected with the same volume of normal saline. After modeling, the rats in the low-, medium- and high-dose XYF groups were treated intragastrically with XYF at 10.4, 20.8 and 41.6 g/kg/d, bid, respectively, those in the normal and model control groups with the same volume of distilled water, and those in the TP group intramuscularly with TP at 5.21 mg/kg/d, qd alt, all for 28 days. After treatment, the supernatant was obtained for measurement of the serum T level by ELISA, and the testis tissue collected for determination of the mRNA and protein expressions of BMAL1, NR1D1, PER2, CRY1, StAR and CYP11A1 by RT-qPCR and Western blot. RESULTS: Compared with the normal controls, the rats in the LOH model control group showed significantly decreased serum T and mRNA and protein expressions of BMAL1, NR1D1, PER2, CRY1, StAR and CYP11A1 (P < 0.05). In comparison with the findings in the model controls, the T level was remarkably increased in the TP and XYF groups (P < 0.05), the expressions of StAR mRNA and CYP11A1 mRNA and protein markedly up-regulated in the high-dose XYF group (P < 0.05), and so was the expression of the StAR protein in the XYF and TP groups (P < 0.05), those of BMAL1 and NR1D1 proteins and PER2 mRNA and protein in the high-dose XYF group (P < 0.05), those of BMAL1 mRNA and CRY1 protein in the medium- and high-dose XYF groups (P < 0.05), that of NR1D1 mRNA in the XYF and TP groups (P < 0.05), and that of CRY1 mRNA in the medium- and high-dose XYF and TP groups (P < 0.05). CONCLUSION: Xiongcan Yishen Formula could up-regulate the expressions of the clock genes in the testis tissue of the LOH rats and increase the serum T level as well, which may underlie the mechanisms of Xiongcan Yishen Formula acting on LOH.

[Potential mechanisms of Jiarong Tablets in the treatment of late-onset hypogonadism in men: An exploration based on network pharmacology].

OBJECTIVE: To study the therapeutic targets and related signaling pathways of Jiarong Tablets (JRT) in the treatment of late-onset hypogonadism (LOH) in males by network pharmacology, and further analyze its potential action mechanism. METHODS: Using the Chinese Medicine System Pharmacology Analysis Platform (TCMSP), we obtained the active ingredients and therapeutic targets of JRT, disease targets of LOH through the GeneCards and OMIM databases, and drug-disease common targets, followed by drawing a Vennny's diagram of the common targets. We constructed a protein-protein interaction (PPI) network of the common targets using STRING and an intersection network of JRT active ingredients-LOH-targets with Cytoscape 3.7.2, performed GO bio-functional and KEGG enrichment analyses of the common targets using the R-Language software, and identified the potential signaling pathways of JRT acting on LOH. RESULTS: Totally, we obtained 80 bioactive ingredients from JRT and 64 common targets of LOH, with IL-6, INS, AKT1, JUN and MAPK8 as the core targets in the order of the frequency occurrences. GO and KEGG analyses showed that these targets mainly involved the MAPK, HIF-1, Ras and ErbB signaling pathways. CONCLUSION: JRT acts on LOH with multiple targets, through multiple routes and at multiple levels, which is related to the expression of testosterone synthetase, oxidative stress and apoptosis of Leydig cells.

[Establishment and evaluation of a rat model of late-on-set hypogonadism using D-galactose].

OBJECTIVE: To explore the feasibility and duration of establishing a model of late-on-set hypogonadism (LOH) in rats using D-galactose (D-gal) and the optimal concentration of D-gal in modeling. METHODS: Thirty-five 8-week-old male SD rats were randomly divided into seven groups of an equal number to receive intraperitoneal injection of normal saline (normal control) or D-gal at 480 mg/kg/d (high-dose), 240 mg/kg/d (medium-dose) and 120 mg/kg/d (low-dose) for 6 or 8 weeks respectively. Another five 18-month-old SD rats were taken as positive controls. After modeling, the animals were subjected to tail suspension test and mating test, weighed, and then sacrificed for examination of the levels of fasting blood glucose (FBG) and serum testosterone (T), testis index and semen parameters, determination of the expressions of the BMAL1 and NR1D1 proteins in the testis tissue by Western blot, and observation of the pathological and ultrastructural changes in the testis tissue under the light microscope and transmission electron microscope. RESULTS: Compared with the normal controls, the serum T level was significantly decreased in the 6-week medium-dose D-gal (6WMD), 6-week high-dose D-gal (6WHD), 8WMD, 8WHD and positive control groups (P < 0.05), and so were sperm concentration and the percentage of progressively motile sperm (PMS) in the 8WHD and positive control groups (P < 0.05), and the numbers of captures and ejaculations in all the model rats and positive controls (P < 0.05), while the body weight was markedly increased in the 8-week low-dose D-gal (8WLD), 8WMD and 8WHD groups (P < 0.05), and so were the tail suspension time in the 6WHD, 8WHD, 8WMD, 8WLD and positive control groups (P < 0.05) and the FBG level in all the model rats and positive controls (P < 0.05). The expression of the BMAL1 protein was significantly decreased in the 6WHD, 8WLD, 8WMD, 8WHD and positive control groups (P < 0.05), and so was that of NR1D1 in the 8WMD, 8WHD and positive control groups (P < 0.05). Compared with the positive controls, no statistically significant differences were observed in the serum T level in the 6WHD and 8WHD groups (P > 0.05), the testis index in the 8WMD and 8WHD groups (P > 0.05), sperm concentration, percentage of FMS, time of tail suspension and numbers of captures and ejaculations in the 8WHD group (P > 0.05), the level of FBG in the 6WMD, 6WHD, 8WLD, 8WMD and 8WHD groups (P > 0.05), the expression of BMAL1 in the 6WHD, 8WLD, 8WMD and 8WHD groups (P > 0.05), and that of NR1D1 in the 6WLD, 8WMD and 8WHD groups (P > 0.05). Pathological and ultrastructural changes in the testis tissue were observed in all the model rats and positive controls, most significantly in the 8WHD and positive control groups. CONCLUSION: It is feasible to establish an LOH model in male SD rats by intraperitoneal injection of D-gal, most recommendably at the centration of 480 mg/kg/d for 8 weeks.

[Establishment and evaluation of an AAPH-induced oxidative stress model in mouse Leydig cells in vitro].

OBJECTIVE: To establish a model of oxidative stress (OS) injury in mouse Leydig cells using α-α'-azodiisobutyramidine hydrochloride (AAPH) and evaluate the physiological function. METHODS: In Experiment 1, we treated mouse TM3 Leydig cells with AAPH at 0, 1, 5, 10, 50 and 100 mmol/L for 4, 8 and 24 h respectively and measured the activity of the cells using MTS, their aging by ß-galactoside staining, mitochondrial membrane potential by JC-1 fluorescence and mitochondrial DNA copy number by qPCR. In Experiment 2, we treated the TM3 cells selected in Experiment 1 according to the AAPH concentration range (≤10 mmol/L) with AAPH at 0, 0.1, 0.5, 1, 2, 4, 6, 8 and 10 mmol/L for 24, 48 and 72 h respectively, detected the activity and aging of the cells and the ROS positive rate, and determined the optimal concentration and time of AAPH in inducing OS injury in the TM3 cells. RESULTS: Experiment 1 showed that the survival rate of the TM3 cells was ≥50% in the 4-h 50 mmol/L, 8-h 10 mmol/L and 24-h 5 mmol/L AAPH groups, the initial concentration of AAPH was ≤10 mmol/L, with the action time of ≥24 h. Experiment 2 manifested that in the 24-h 6 mmol/L AAPH group, the survival rate of the TM3 cells was ≥70%, with an ROS positive rate of 56.88%, normal mitochondrial membrane potential, increased number of mtDNA copies, but no senescence. CONCLUSIONS: Treatment with AAPH at the concentration of 6 mmol/L for 24 hours is suitable for induction of OS injury in the TM3 cells.

[Improving effect of Jiarong Tablets on the morphological structure and function of the testis in rats with late-onset hypogonadism].

OBJECTIVE: To investigate the effect of Jiarong Tablets (JRT) on the testicular morphology and function of rats with late-onset hypogonadism (LOH). METHODS: LOH models were established in 8 eighteen-month-old male SD rats, treated intragastrically with distilled water (the model control group, n = 4) or JRT at 0.375 g/kg/d, qd (the JRT group, n = 4), and another 5 two-month-old normal male SD rats were also given distilled water by gavage (normal control group), all for 28 days. Then all the rats were weighed and sacrificed for measurement of the serum T level and pathological and electron microscopic examination of the testis tissue. RESULTS: Compared with the normal controls, the LOH models showed significantly decreased testis coefficient (P < 0.05) and serum T level (ï¼»3.40 ± 0.06ï¼½ vs ï¼»5.88 ± 0.46ï¼½ ng /ml, P < 0.05). No statistically significant differences were observed in the model control and JRT groups in the body weight and testis coefficient (P > 0.05), but the serum T level (ï¼»4.50 ± 0.78ï¼½ ng/ml) was remarkably decreased in the latter (P < 0.05). In comparison with the model controls, the rats treated with JRT exhibited increases in the sperm count in the seminiferous tubules and the amount of testicular interstitial cells. Electron microscopy revealed a markedly increased number of mitochondria in the JRT-treated animals, with some mitochondrial sheaths and cristae but no obvious mitochondrial edema. CONCLUSIONS: Jiarong Tablets can elevate the serum T level and improve the testicular morphology and ultrastructure of LOH rats.

[Xiongcan Yishen Prescription up-regulates the expressions of cholesterol transport proteins, steroidogenic enzymes and SF-1 in the Leydig cells of rats with late-onset hypogonadism].

OBJECTIVE: To investigate the effects of Xiongcan Yishen Prescription (XYP) on the expressions of cholesterol transport proteins, steroidogenic enzymes and steroidogenic factor-1 (SF-1) in the Leydig cells of the rats with late-onset hypogonadism (LOH). METHODS: Twenty-five 18-month-old male SD rats were randomly divided into five groups of equal number, LOH model control, testosterone propionate (TP) and low-, medium- and high-dose XYP, and another 5 two-month-old male SD rats included as normal controls. After modeling, the animals in the TP group were treated by intramuscular injection of TP at 5.21 mg/kg qd alt, those in the low-, medium- and high-dose XYP groups intragastrically with XYP at 10.4, 20.8 and 41.6 g/kg qd alt respectively, and those in the LOH model and normal control groups with saline, all for 28 successive days. Then, all the rats were sacrificed for determination of the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the Leydig cells by Western blot. RESULTS: The expressions of StAR, TSPO, CYP11A1, HSD3B7, HSD17B4 and SF-1 in the Leydig cells were significantly decreased in the LOH model controls compared with those in the normal controls (P< 0.05), but remarkably increased in the low-, medium- and high-dose XYP groups in comparison with those in the LOH model control group (P< 0.05). CONCLUSIONS: Xiongcan Yishen Prescription can up-regulate the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the rat Leydig cells, which might be one of the possible mechanisms of the prescription in the treatment of LOH.

[Xiongcan Yishen Prescription upregulates the expressions of eNOS and cGMP in the penile tissue of ED rats with liver depression and kidney deficiency].

OBJECTIVE: To investigate the effect of Xiongcan Yishen Prescription (XYP) on the expressions of eNOS and cGMP in the penile tissue of ED rats with liver depression and kidney deficiency (LDKD). METHODS: The model of ED-LDKD was established in 30 eight-week-old SPF-class male SD rats by injecting hydrocortisone intramuscularly and binding the limbs for 14 days, and another 10 rats were taken as blank controls. Then, the model rats were randomized into six groups of equal number and treated intragastrically with distilled water (model control), tadalafil tablets at 0.52 mg/kg/d (tadalafil control), Shugan Yiyang Capsules 0.3125 g/kg/d (SYC control), and XYP at 10.4 g/kg/d (low-dose XYP), 20.8 g/kg/d (medium-dose XYP) and 41.6 g/kg/d (high-dose XYP), bid, for 28 successive days, respectively. Before and after modeling and after 28-day treatment, the animals were subjected to tail suspension and mating tests. The next day after medication, the penile tissues of the rats were harvested for determining the expression levels of eNOS and cGMP proteins by immunohistochemical analysis of the mean optical density. RESULTS: Compared with the model controls, the rats of the high-, medium- and low-dose XYP and SYC control groups all showed significant decreases in the tail suspension time (ï¼»3.17 ± 0.11ï¼½ vs ï¼»2.58 ± 0.25ï¼½, ï¼»2.52 ± 0.31ï¼½, ï¼»2.51 ± 0.3ï¼½ and ï¼»2.57 ± 0.29ï¼½ min, P < 0.05) and mount latency (ML) (ï¼»9.23 ± 0.11ï¼½ vs ï¼»1.21 ± 0.12ï¼½, ï¼»2.17 ± 0.16ï¼½, ï¼»2.26 ± 0.13ï¼½, ï¼»1.23 ± 0.15ï¼½ and ï¼»2.48 ± 0.18ï¼½ min, P < 0.05) but increases in mount frequency (MF) (ï¼»0.48 ± 0.18ï¼½ vs ï¼»3.29 ± 0.11ï¼½, ï¼»3.18 ± 0.11ï¼½, ï¼»3.05 ± 0.05ï¼½, ï¼»3.23 ± 0.12ï¼½ and ï¼»3.2 ± 0.28ï¼½ times, P < 0.05) and intromission frequency (IF) (ï¼»0.8 ± 0.84ï¼½ vs ï¼»11.8 ± 0.84ï¼½, ï¼»11.2 ± 1.48ï¼½, ï¼»9.4 ± 1.14ï¼½, ï¼»11.4 ± 1.14ï¼½ and ï¼»10 ± 1.22ï¼½ times, P < 0.05). The eNOS and cGMP proteins were mainly expressed in the nucleus and cytoplasm of the arterial and venous endothelial cells and sinusoidal endothelial cells of the cavernous, as brownish yellow particles in a scattered and focal pattern. Both the expressions of eNOS and cGMP in the penile tissue were remarkably upregulated in the high-, medium- and low-dose XYP and SYC control groups as compared with those in the model control (P < 0.05) but exhibited no statistically significant difference between the tadalafil and model control groups (P > 0.05). CONCLUSIONS: Xiongcan Yishen Prescription can relieve the depression symptoms, increase the mount frequency, activate the NO/cGMP pathway, and upregulate the expressions of eNOS and cGMP in the penile tissue of ED rats with liver depression and kidney deficiency.

[Xiongcanyishen Prescription increases mitochondrial autophagy of Leydig cells in LOH rats via PINK1 and Parkin pathways].

OBJECTIVE: To investigate the effects of PINK1 and Parkin pathways mediated by Xiongcanyishen Prescription (XP) on the mitochondrial autophagy of Leydig cells in male rats with late-onset hypogonadism (LOH). METHODS: Twenty 18-month-old male SD rats were randomly divided into an LOH model control, a low-dose XP, a medium-dose XP and a high-dose XP group, and another 5 two-month-old male SD rats were included as normal controls. The animals in the low-, medium- and high-dose XP groups were treated intragastrically with XP granules at 10.4, 20.8 and 41.6 g/kg, while the normal and LOH model controls with the same volume of distilled water, all for 28 successive days. Then the testis tissues of the rats were harvested for observation of the ultrastructure of the Leydig cells under the electron microscope, and the expressions of PINK1, Parkin and p62 proteins were detected by Western blot. RESULTS: The mitochondria in the Leydig cells of the normal controls were basically normal in morphology, with evident autophagy, those of the model controls showed less autophagy, and those in the XP intervention groups all exhibited autophagy. The expressions of PINK1 and Parkin proteins in the testis tissue were significantly lower and that of p62 markedly higher in the LOH model than in the normal controls (P < 0.05). Compared with the rats in the model control group, those treated with XP showed remarkable elevation in the expression of PINK1 in the low-, medium- and high-dose groups and that of Parkin in the medium- and high-dose groups (P < 0.05), but a significantly down-regulated expression of p62 in all the three XP groups (P < 0.05). CONCLUSIONS: Xiongcanyishen Prescription can enhance the decreased mitochondrial autophagy of Leydig cells in LOH rats, which may be related to its ability of up-regulating the expressions of PINK1 and Parkin and down-regulating that of p62 and its influence on the ultrastructure of Leydig cells.

[Quantification of crop residue burned areas based on burning indices using Landsat 8 image].

Crop residue burning leads to atmospheric pollution and is an enormous waste of crop residue resource. Crop residue burning can be monitored timely in large regions as the fire points can be recognized through remotely sensed image via thermal infrared bands. However, the area, the detailed distribution pattern and especially the severity of the burning areas cannot be derived only by the thermal remote sensing approach. The burning index, which was calculated with two or more spectral bands at where the burned and unburned areas have distinct spectral characteristics, is widely used in the forest fire investigation. However its potential application for crop residue burning evaluation has not been explored. With two Landsat 8 images that cover a part of the Songnen Plain, three burning indices, i.e., the normalized burned ratio (NBR), the normalized burned ratio incorporating the thermal band (NBRT), and the burned area index (BAI), were used to classify the crop residue burned and unburned areas. The overall classification accuracies were 91.9%, 92.3%, and 87.8%, respectively. The correlation analysis between the indices and the crop residue coverage indicated that the NBR and NBRT were positively correlated with the crop residue coverage (R2 = 0.73 and 0.64, respectively) with linear regression models, while the BAI was exponentially correlated with the crop residue coverage (R2 = 0.68). The results indicated that the use of burning indices in crop residue burning monitoring could quantify crop residue burning severity and provide valuable data for evaluating atmospheric pollution.