Inhibitory effect of bushen huoxue formula against dehydroepiandrosterone-induced inflammation in granulosa cells through TLR4/NF-κB signaling pathway.

Androgen exposure may be an important factor in promoting the development of polycystic ovary syndrome (PCOS) and disease progression. Bushen Huoxue Formula (BHF), a traditional Chinese medicine, is prescribed in clinical settings as a PCOS remedy, albeit with unclear pharmacological effects on granulosa cells. The present research explores potentially advantageous BHF impacts and whereby BHF alleviates dehydroepiandrosterone (DHEA)-induced inflammation and endocrine disruption. Six chemical components in BHF were identified and fingerprint analysis showed good reproducibility. Using a human granulosa cell line (KGN), BHF effects on cell viability, secretion of steroidogenic and inflammatory factors were evaluated and TLR4/NF-κB pathway expression was examined. Our results demonstrate that BHF treatment of KGN cells in a DHEA-induced inflammatory state led to increased cell viability, decreased testosterone and estradiol production, and decreased CYP19A1 and HSD3B2 mRNA expression. Further experiments revealed that BHF inhibited the expression of pro-inflammatory cytokines and considerably hindered up-regulation in protein levels of TLR4, MyD88, and TRAF6, while inhibiting the activation of NF-κB and phosphorylation of IκBα. Collectively, BHF administration protected granulosa cells from DHEA-induced injuries through down-regulating pro-inflammatory cytokines and blocking the pathway of TLR4/NF-κB. Therefore, BHF hold promise as a therapeutic formulation for preventing androgen induced PCOS.

Phytosterols in mountain cultivated ginseng, as master healthy ageing dietary supplement, activates steroid signaling in ageing Drosophila melanogaster.

Mountain cultivated ginseng (MCG) is planted in mountain forests to simulate traditional wild ginseng; therefore, it has a greater pharmacological effect than cultivated ginseng (CG) in the garden; however, insufficient evidence confirms this theory. In light of the health-promoting and life-extending properties of ginseng, we analyzed the efficacy of MCG and CG. Initial observations revealed that the phytosterols content of MCG was higher than that of CG, with a positive correlation to the duration of growth. The distinction between phytosterols in MCG and in CG is predominately determined by the stigmasterol content using High-Performance Liquid Chromatography (HPLC). The lifespan of Drosophila melanogaster (fruit flies) that aged naturally was prolonged by phytosterols in MCG and CG and stigmasterols. Further, they prolonged healthy ageing as measured by progeny numbers, length of sleep, climbing distance, and survival following oxidative damage. The findings of behavioral observations revealed that phytosterols in MCG were more efficacious than in CG in promoting health maintenance and life extension; moreover, stigmasterol indicated that these effects were dose-dependent. Stigmasterols, phytosterols in MCG and CG have restored age-associated decreases in steroid hormone levels. Notably, molecular docking was predicted to promote stigmasterol's binding to the steroid hormone receptor ECR due to its similarity to steroid hormones. In addition, stigmasterols triggered the steroid hormone signaling pathway by increasing the activity of key genes Eip75B and Br in 20E signaling and Jhamt, HmGR, Met, and Kr-h1 in JH signaling. Phytosterols, as a natural product, regulated health and longevity as a dietary supplement similar to that of steroids, which supported the social requirements of healthy ageing.

New insights into ginsenoside Rg1 regulating the niche to inhibit age-induced germline stem cells depletion through targeting ECR/BMP signaling pathway in Drosophila.

PURPOSE: The age-induced imbalance in ecological niches leads to the loss of GSCs, which is the main reason for ovarian germline senescence. Ginsenoside Rg1 can delay ovarian senescence. Here, we shed light on new insights of ginsenoside Rg1 in regulating the niche to maintain GSCs self-renewal and discussing related molecular mechanisms. METHODS: The differences among GSC number, reproductive capacity of naturally aging female Drosophila after ginsenoside Rg1 feeding were analyzed by immunofluorescence and behavior monitoring. The expressions of the active factors in the niche and the BMP signaling were analyzed through Western blot and RT-qPCR. The target effect was verified in the ECR mutant and combined with the molecular docking. RESULTS: Ginsenoside Rg1 inhibited the age-induced reduction of the GSCs number and restored offspring production and development. Ginsenoside Rg1 promoted the expression of anchor proteins E-cadherin, stemness maintenance factor Nos and differentiation promoting factor Bam, thereby GSCs niche homeostasis was regulated. In addition, ginsenoside Rg1 was bound to the LBD region of the hormone receptor ECR. Ginsenoside Rg1 promotes the regeneration of GSCs by targeting the ECR to increase pSmad1/5/8 expression and thereby activating the BMP signaling pathway. In addition, ginsenoside Rg1 maintenance of niche homeostasis to promote GSCs regeneration is dependent on ECR as demonstrated in ECR mutants. CONCLUSIONS: Ginsenoside Rg1 regulated the ecological niche homeostasis of GSCs and promoted the regeneration of GSCs by targeting the ECR/BMP signaling pathway in hormone-deficient states in aging ovaries. It is of great significance for prolonging fertility potential and delaying ovarian senescence.

Comparison of the metabolomic and proteomic profiles associated with triterpene and phytosterol accumulation between wild and cultivated ginseng.

Wild ginseng is thought to be superior in its medicinal quality to cultivated ginseng, potentially owing to the differences in active components. This study was designed accordingly to assess the differences in secondary metabolite components and their synthesis in wild and cultivated ginseng by using quantitative proteomics combined with secondary metabolomics approaches. A total of 72 secondary metabolites were found to be differentially abundant, of which dominant abundant in wild ginseng primarily included triterpenoid saponins (ginsenosides) and phytosterols. Ginsenoside diversity was increased in wild ginseng, particularly with respect to rare ginsenosides. Ginsenoside Rk1, F1, Rg5, Rh1, PPT, Rh2, and CK enriched in wild ginseng were validated by HPLC. In addition to ginsenosides, stigmasterol and ß-sitosterol were accumulated in wild ginseng. 102 differentially expressed proteins between wild and cultivated ginseng were identified using iTRAQ labeling technique. Among them, 25 were related to secondary metabolism, mainly involved in sesquiterpene and triterpene biosynthesis, which was consistent with metabolomics results. Consistently, the activity levels of HMGR, FDPS, SS, SE, DS, CYP450, GT and CAS, which are key enzymes related to ginsenoside and phytosterol biosynthesis, were confirmed to be elevated in wild ginseng.The biosynthesis of ginsenosides and phytosterols in wild ginseng is higher than that in cultivated ginseng, which may be related to natural growth without artificial domestication. To some extent, this study explained the accumulation of pharmacodynamic components and overall quality of ginseng, which could provide reference for the germplasm improvement and planting of ginseng.

CDH1 promoter polymorphism and stomach cancer susceptibility.

The relationship of stomach cancer susceptibility and the presence of E-cadherin (CDH1) promoter -160 C/A polymorphism had been reported with conflicting results. To further explore the association of this polymorphism with stomach cancer susceptibility, we performed an extensive search of relevant studies and carried out a meta-analysis to obtain a more precise estimate. A total of 16 studies including 2,611 cases and 3,788 controls were involved in this meta-analysis. When all studies involved, the meta-analysis results suggest no statistically significant association between CDH1 -160 C/A polymorphism and stomach cancer risk (CA vs. CC: OR = 1.01, 95% CI: 0.85-1.19; AA vs. CC: OR = 1.05, 95% CI: 0.75-1.46; dominant model: OR = 1.02, 95% CI: 0.86-1.20; recessive model: OR = 1.04, 95% CI: 0.76-1.41). When subgroup analyses were performed by ethnicity, the A-allele carriers conferred a decreased stomach cancer risk in Asians (AA vs. CC: OR = 0.67, 95% CI: 0.47-0.96; dominant model: OR = 0.85, 95% CI: 0.72-0.99), but no statistically significant association was found in Caucasians. In conclusion, this meta-analysis suggests that CDH1 -160 A-allele may play a protective role of stomach cancer development in Asians but not in Caucasians.

Ginsenosides improve reproductive capability of aged female Drosophila through mechanism dependent on ecdysteroid receptor (ECR) and steroid signaling pathway.

Aging ovaries caused diminished fertility and depleted steroid hormone level. Ginsenosides, the active ingredient in ginseng, had estrogen-like hormonal effects. Although ginsenosides were well known for their ability to alleviate many age-related degenerative diseases, the effect of ginsenosides on the decline in reproductive capability caused by aging, as well as the mechanism, are unknown. We found that ginsenosides improved the quantity and quality of the offspring, prolonged life and restored muscle ability in aged female Drosophila. In addition, ginsenosides inhibited ovarian atrophy and maintained steroid hormone 20-Hydroxyecdysone (20E) and juvenile-preserving hormone (JH)) levels. Ginsenosides activated ecdysteroid receptor (ECR) and increased the expression of the early transcription genes E74 and Broad (Br), which triggered steroid signaling pathway. Meanwhile, ginsenosides promoted JH biosynthesis by increasing the expression of Hydroxyl-methylglutaryl-CoA reductase (HMGR) and juvenile hormone acid O-methyltransferase (JHAMT). Subsequently, JH was bound to Methoprene Tolerant (Met) and activated the transcription of the responsive gene Kruppel Homolog 1 (Kr-h1), which coordinated with 20E signaling to promote the reproduction of aged female Drosophila. The reproductive capacity and steroid hormone levels were not improved and the steroid signaling pathway was not activated in ginsenoside-treated ECR knockout Drosophila. This suggested that ginsenosides played a role dependent on targeted ECR. Furthermore, 17 kinds of ginsenoside monomers were identified from the total ginsenosides. Among them, Rg1, Re and Rb1 improved the reproductive capacity and steroid hormone levels of aged female Drosophila, which has similar effects to the total ginsenoside. These results indicated that ginsenosides could enhance the reproductive capacity of aged female Drosophila by activating steroid signals dependent on nuclear receptor ECR. In addition, ginsenoside monomers Rg1, Rb1 and Re are the main active components of total ginsenosides to improve reproductive ability. This will provide strong evidence that ginsenosides had the potential to alleviate age-induced reproductive degradation.

Protective effects of phenolic acid extract from ginseng on vascular endothelial cell injury induced by palmitate via activation of PI3K/Akt/eNOS pathway.

Elevated free fatty acids may impair insulin-mediated signaling to eNOS that contributes to the pathophysiology of endothelial dysfunction. Previous studies have indicated the protective effect of ginseng and the regulatory potential of phenolic acid components from other plants on endothelial function. Therefore, this study investigated the protective effects of phenolic acid extract from ginseng (PG2) on endothelial cells against palmitate-induced damage. We found that PG2 increases cell viability, inhibits the palmitate-induced intracellular accumulation of lipids, and the overexpression of endothelin-1 (ET-1) through enhancing the phosphorylation of the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway. The results of this study may be valuable for the development of PG2 to combat the endothelial cell damage caused by hyperlipidemia. PRACTICAL APPLICATION: We proved that phenolic acid extract from ginseng has a protective effect on free fatty acid-induced endothelial dysfunction in vitro. This study provides experimental data for the application of ginseng-derived phenolic acids in treating cardiovascular disease.

Traditional Chinese medicine Kang Xian Fu Fang I is effective for prophylaxis and treatment of alcoholic liver disease in rats.

BACKGROUND: Reversal of liver fibrosis is one of the key steps in the prevention and treatment of alcoholic liver disease (ALD), but the mechanism is unknown. This study was to investigate the effects of the Chinese medicine Kang Xian Fu Fang I (KXI) on prophylaxis and treatment of ALD in rats and its possible mechanism of action. METHODS: Eighty male Wistar rats were randomly divided into four groups: normal control; ALD model; treatment of ALD with KXI; and prophylaxis of ALD by KXI. At the end of 4, 8, 12 and 16 weeks, five rats from each group were anesthetized and their livers were removed for pathological studies using hematoxylin-eosin and Masson stain, immunohistochemical studies, and flow cytometry for matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Blood samples were taken for hyaluronic acid (HA) assay. RESULTS: Serum HA level and liver collagen content were lower in the groups given KXI for prophylaxis and treatment than in ALD model group (P<0.05). The levels of MMP-2 and MMP-9 were also decreased in the prophylaxis and treatment groups (P<0.05). Immunohistochemistry showed immunoreactive MMP-2 in endothelial cells of the hepatic artery and portal vein, sinusoidal endothelial cells, and sinusoidal cells. Immunoreactive MMP-9 occurred in the hepatic cells around the veins and sinusoidal cells. CONCLUSIONS: KXI can effectively inhibit or reverse the course of ALD. This may be attributable to its capacity to inhibit the expression of MMP-2 and MMP-9.

Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions.

AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining. RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation. CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.

Effects of ethanol on liver sinusoidal endothelial cells-fenestrae of rats.

BACKGROUND: Important advances have been made in research into the mechanism of alcoholic liver disease (ALD) over the past few years, but the role of liver sinusoidal endothelial cell (LSEC) in ALD has not been elucidated adequately. This study was undertaken to investigate the effect of ethanol on fenestrae of LSECs in rats. METHODS: A rat model of alcoholic liver disease was established by means of direct intragastric instillation of ethanol. Fifty-five rats of experimental (35 rats) and control (20) groups were sacrificed at the end of 4, 8, 12 weeks respectively, and also at the end of 12-week abstinence. After heart perfusion, the liver tissue was fixed and stained with hematoxylin and eosin for observation of serial changes of LSEC-fenestrae under a transmission electron microscope. RESULTS: Normal LESC was flat with a nucleus and organelles arranged regularly. The distal cytoplasm displayed as a lamina with many fenestrae, lacking the basement membrane(BM) underneath the endothelium. At the end of 4-week alcohol feeding, the number of fenestrae decreased at the distal cytoplasm in some LSECs, without the formation of the BM underneath the endothelium. At the end of 8 weeks, the number of fenestrae decreased significantly or even disappeared. The BM began to develop incompletely underneath the endothelium, while the active fibroblast appeared. At the end of 12 weeks, the number of fenestrae decreased more significantly and the complete BM could even be seen. But the changes were mostly limited in the single or adjoining sinus, and fibrosis was scarcely formed. At the end of 12-week abstinence, defenestration and formation of the endothelial BM lightened significantly. CONCLUSIONS: Defenestration and formation of the BM in LSECs develop gradually with the chronic stimulation of ethanol. Hepatic sinusoidal capillarization and fibrosis will be seen if their state is more serious. These early changes, i.e., limited and regional defenestration and capillarization may be the basis of alcoholic peri-fibrosis. This kind of hepatic fibrosis is reversible after removal of etiological factors.

[Deletion of p15 and pl6 genes and overexpression of STK15 gene in primary hepatocellular carcinoma].

OBJECTIVE: To investigate the association of p15 and pl6 genes deletion and STKI5 gene overexpression in primary hepatocellular carcinoma (PHC). METHODS: The carcinoma tissue and the adjacent normal tissue were taken from 30 PHC patients during operations who had had neither chemotherapy nor radiotherapy preoperatively. DNA was extracted from the tissues and PCR was used to determine the homozygous deletion of p15 exon2 (pl5E2) and pl6 exon 2 (pl6E2). RNA was extracted, cDNA was synthesized by RT-PCR, and the expression of STKI5 gene was tested by PCR. Beta-actin was used as an internal control. Average density value (ADV) of STK15 gene and that of beta-actin gene were determined in both carcinoma tissue and the adjacent normal tissue. RESULTS: The rate of p15E2 deletion was 13.3% (4/30) and the rate of p16E2 deletion was 16.7% (5/30) in the carcinoma tissue. The p15E2 and pl6E2 co-deletion rate was 6.7% (2/30). In 19 of the 30 cases (63.3%) the expression of STK15 gene in carcinoma tissue was higher than that in the adjacent normal tissue. The ratio of ADV of STK15 gene to ADV of beta-actin gene (1.53+/-0.31) in the carcinoma tissue was significantly higher than that (0.91+/-0.25) in the paired adjacent normal tissue (t = 2.86). CONCLUSION: The homozygous deletion of p15E2 and p16E2 and overexpression of STKI5 gene may play a role in the oncogenesis and malignant progression of PHC.

[Relationship between interleukin-1B and interleukin-1 receptor antagonist gene polymorphisms and susceptibility to gastric cancer].

OBJECTIVE: To investigate the polymorphisms of interleukin-1B (IL-1B) promotor region 31 C/T and interleukin-1 receptor antagonist (IL-1RN) gene and the relationship between the genotype of IL-1 and IL-1RN and susceptibility to gastric cancer in Chinese population. METHODS: Genomic DNA was extracted from the juxta-cancerous normal mucosal biopsies of 50 patients with gastric cancer taken by endoscopy and from the peripheral lymphocytes of 50 normal controls and then was subjected to PCR amplification. The PCR product was digested by restriction endonuclease Alu I. The product of digestion was subjected to 2% agarose gel electrophoresis and ethidium bromide staining. RESULTS: The IL-1B -31C allele was detected in 75% of normal controls and 53% of patients, while the IL-1B -31T allele was detected in 25% of normal controls and 47% of patients. The frequency of -31C/-31C was 56% (28/56) and 30% (15/50) in normal controls and patients respectively. The frequency of -31C/-31T was 38% (19/56) and 46% (23/50) in normal controls and patients respectively. The frequency of -31T/-31T was 6% (3/56) and 24% (12/50) in normal controls and patients respectively. IL-1B -31T carriers were at increased risk of gastric cancer with an odds ratio of 7.5 for -31T/-31T homozygotes (95% CI, 2.0 approximately 27.6) and of 2.3 for -31C/-31T (95% CI, 0.9 approximately 5.4) respectively. Only three of the five kinds of polymorphism of IL-1RN (1/1, 1/2 and 2/2) were found in this study. The frequency of 1/1 was 54% (27/50) and 24% (12/50) in normal controls and patients respectively. The frequency of 1/2 was 38% (19/50) and 50% (25/50) in normal controls and patients respectively. The frequency of 2/2 was 8% (4/50) and 26% (13/50) in normal controls and patients respectively. The IL-1RN * 1 allele was detected in 73% of normal controls and 49% of patients; while the IL-1RN * 2 allele was detected in 27% of normal controls and 51% of patients. IL-RN * 2 carriers were at increased risk of gastric cancer with an odds ratio of 7.31 for 2/2 homozygotes (95% CI 2.13 approximately 25.09) and of 2.96for 1/2 heterozygotes (95% CI 1.22 approximately 7.21), respectively. CONCLUSION: The polymorphism of promotor region -31C/T of IL-1B gene and the polymorphism of IL-1RN genes 1/2 and 2/2 are associated with the susceptibility of gastric cancer in Chinese. Carrying -31T allele increases the risk of gastric cancer. Polymorphism of IL-1RN and IL-1B gene may be used as indicators of susceptibility of gastric carcinogenesis.

[The influence of alcohol on the liver sinusoids endothelial cell fenestrae of rats].

OBJECTIVE: To study the influence of alcohol on the liver sinusoids endothelial cell (LSEC) fenestrae of rats. METHODS: Setting up the rat model of alcoholic liver disease by orogastric administration of alcohol, then kill the experimental and control groups of rats at the end of 4 weeks, 8 weeks and 12 weeks after alcohol feeding, and also at the end of another 12 weeks after balance foods feeding succeeding with alcohol feeding for 12 weeks. Staining the liver tissue by means of HE method and observing the successive change of LSEC fenestrae by transmission electron microscope. RESULTS: The normal LSEC was flat with nucleus and organelle arranged regularly. The distal cytoplasm displayed as lamina with many fenestrae, not accompanied by basement membrane (BM) formation under the endothelial cell. At the end of 4 weeks of alcohol feeding, fenestrae decreased at the partial distal LSEC cytoplasm, but no BM developed. At the end of 8 weeks, fenestrae decreased significantly, even disappeared, with the BM developed incompletely under the endothelial cell. Concomitantly, fibroblast with active function developed. At the end of 12 weeks, the changes became more obvious; the complete BM could even be seen. However, this kind of changes was mostly limited in the single or adjoining sinusoids, as well as with little widespread formation of fibrosis. At the end of 12 weeks of stopping alcohol feeding, defenestrae and development of BM attenuated obviously. CONCLUSION: The defenestrae and BM of LSEC develop gradually with the chronic alcohol stimulation. Sinusoid capillarization and liver fibrosis even form when significant changes happen. The early change of the limited defenestrae and capillarization may be the basis of alcohol periportal fibrosis formation. This kind of liver fibrosis can be reversible after stopping alcohol feeding.