Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. AIM OF THE STUDY: This research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). MATERIALS AND METHODS: Primary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3-7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. RESULTS: In ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. CONCLUSIONS: These results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

Feedback mechanisms of periphytic biofilms to ZnO nanoparticles toxicity at different phosphorus levels.

The increasing use of nanoparticles (NPs) has raised concerns about their potential environmental risks. Many researches on NPs focused on the toxicity mechanism to microorganisms, but neglect the toxicity effects in relation to nutritional conditions. Here, we evaluated the interactive effects of zinc oxide (ZnO) NPs and phosphorus (P) levels on the bacterial community and functioning of periphytic biofilms. Results showed that long-term exposure to ZnO NPs significantly reduced alkaline phosphatase activity (APA) of periphytic biofilms just in P-limited conditions. Co-occurrence network analysis indicated that ZnO NPs exposure reduced network complexity between bacterial taxa in P-limited conditions, while the opposite trend was observed in P-replete conditions. Correlation analysis and random forest modeling suggested that excessive Zn2+ released and high reactive oxygen species (ROS) production might be mainly responsible for the inhibition of APA induced by ZnO NPs under P-limited conditions, while adjustment of bacterial diversity and improvement of keystone taxa cooperation were the main mechanisms in maintaining APA when subjected to weak toxicity of ZnO NPs in P-replete conditions. Taken together, our results provide insights into the biological feedback mechanism involved in ZnO NPs exposure on the ecological function of periphytic biofilms in different P nutritional conditions.

Comprehensive evaluation of the combined extracts of Epimedii Folium and Ligustri Lucidi Fructus for PMOP in ovariectomized rats based on MLP-ANN methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney deficiency is the main pathogenesis of osteoporosis based on the theory of "kidney governing bones" in traditional Chinese medicine (TCM). Osteoporosis is a systemic disease; kidney deficiency influences the growth, aging and reproduction of human body, reflecting in endocrine, nerve, immunity, metabolism and other functions. Multi-target drugs composed of natural non-toxic products from kidney-reinforcing herbs, are being investigated for the treatment of osteoporosis. Therefore, it is necessary and imperative to develop an objective and comprehensive method to evaluate and compare the effects of herbs with listed drugs. AIM OF THE STUDY: This study was designed to evaluate and compare the therapeutic effects and the underlying molecular mechanism of the combined extracts of Epimedii Folium and Ligustri Lucidi Fructus (EL) with Raloxifene hydrochloride (RH) in ovariectomy (OVX)-induced postmenopausal osteoporosis (PMOP) rats based on the multi-layer perception (MLP)-artificial neural network (ANN) model. MATERIALS AND METHODS: Female SD rats were subjected to either sham surgery (n = 8) or bilateral OVX (n = 48). One week after recovering from surgery, the OVX-induced rats were randomly divided into three groups: OVX model group (n = 32, every 8 rats were killed at the end of the 5th, 9th, 11th or 13th week after OVX), EL group (treated with EL 0.35 g/kg, n = 8), and RH group (treated with RH 6.25 mg/kg, n = 8). The rats in the treatment groups were administrated once a day for 12 weeks, then sacrificed. We observed bone mass and quality, bone remodeling, the function of estrogen and TGF-ß1/Smads pathway in all rats. RESULTS: Both EL and RH could increase bone mineral density, enhance bone strength, relieve bone micro-structure degeneration, re-balance bone remodeling, regulate estrogen dysfunction, and up-regulate TGF-ß1 expression. The evaluation of the MLP-ANN model showed that EL and RH had markedly anti-PMOP effects, and there was no significant difference in the comprehensive evaluation of anti-osteoporosis between the two drugs. However, RH had better effects on bone mass and quality and TGF-ß1/Smads pathway than EL; EL had better effects on estrogen function than RH. CONCLUSION: Combined extracts of Epimedii Folium and Ligustri Lucidi Fructus (EL) exhibited bone-protective effects on PMOP. The MLP-ANN method evaluated the efficacy of drugs more comprehensively, which provided a new direction for the evaluation and comparison of drugs.

Systemic osteoprotective effects of Epimedii Folium and Ligustri Lucidi Fructus in senile osteoporosis rats by promoting the osteoblastogenesis and osteoclastogenesis based on MLP-ANN model.

BACKGROUND: Senile osteoporosis (SOP), which is caused by unbalanced bone remodeling, leads to significant economic and societal burdens globally. The combination of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) serves as a commonly-used prescription for SOP in Traditional Chinese Medicine (TCM). This study aimed to evaluate the osteoprotective effects of EF and LLF in combination on SOP rats based on the constructed multilayer perception (MLP)-artificial neural network (ANN) model. METHODS: 15 month old male Sprague-Dawley rats were administrated with EF, LLF or the combination of EF and LLF (EF&LLF) for 2 months, while 17 month old rats were used as the aging control group. All the rats were anesthetized with 25% ethyl carbamate, then their serum liver and bone tissues were taken. We detected bone mass, bone mineral density (BMD), biomechanics and the microstructure of bone trabecula by micro-CT and H&E staining to evaluate the degree of osteoporosis. Blood lipids and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) and liver pathology were use to assess the side effects of drugs. Levels of alkaline phosphatase (ALP) and Tartrate-resistant acid phosphatase (TRACP) and the ratio of ALP to TRACP both in serum and bone were measured for the evaluation of bone turnover rate. The bone mRNA and protein expression of osteoprotegerin (OPG), nuclear factor-kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), d2 isoform of vacuolar (H+) ATPase (ATP6V0d2), insulin-like growth factor (IGF-1), bone morphogenetic protein-2 (BMP2), M-CSF, Wnt5a, transforming growth factor-ß1 (TGF-ß1) were detected for evaluating bone metabolism. RESULTS: The results showed that EF&LLF improved bone mass and bone quality by preventing bone loss, increasing maximal load as well as protecting the micro-structural retrogressive change of trabecular bone in SOP rats; ameliorated the steatosis in the liver and decreased blood lipids and serum ALT, AST and GGT; enhanced bone remodeling by stimulating the expression of ALP and TRACP. At the molecular levels, EF&LLF stimulated the osteoclastogenesis by upregulating the protein and mRNA expression of OPG, RANKL, M-CSF and ATP6V0d2; meanwhile, EF&LLF stimulated osteoblastogenesis by enhancing the expression of TGF-ß1, BMP2, Wnt5a and IGF-1. According to our established MLP model, EF&LLF has a better effect on osteoclastogenesis or steoblastogenesis in SOP rats than EF or LLF. CONCLUSIONS: These findings demonstrate that the systemic bone protective effects of EF&LLF by promoting bone remodeling in aging rats might be a substitute medicine for the treatment of SOP.

Combined Extracts of Epimedii Folium and Ligustri Lucidi Fructus with Budesonide Attenuate Airway Remodeling in the Asthmatic Rats by Regulating Apoptosis and Autophagy.

This study aimed to investigate the effects of the coadministration of budesonide (Bud) and the extracts of Epimedii Folium and Ligustri Lucidi Fructus (EEL) on regulating apoptosis and autophagy in asthmatic rats. Forty Sprague-Dawley rats were divided randomly into five groups (8 rats in each group): normal control (control), asthma model (asthma), Bud (1 mg Bud suspension in 50 ml sterile physiological saline for 30 min), EEL (100 mg/kg EEL), and group of coadministration of Bud and EEL (Bud&EEL, 100 mg/kg EEL plus Bud by nebulized inhalation for 30 min). Rats were sensitized and challenged with ovalbumin for 7 weeks and treated with corresponding drug for 4 weeks. We anesthetized all rats with 25% ethyl carbamate (4 ml/kg) and took lung tissues and BALF after final ovalbumin challenge to observe the lung histopathology and morphometry; apoptosis in BALF and lung tissue; protein expressions of Ki-67, α-SMA, cleaved Caspase-3, p-mTOR, and LC3; and protein and mRNA expressions of Bax, Bcl-2, Caspase-3, P53, mTOR, and Beclin-1. Results showed that Bud&EEL could alleviate airway remodeling, inhibit cell proliferation and autophagy in lung tissue, and promote apoptosis in BALF and lung tissue in ovalbumin-induced asthma rats through downregulating the protein expressions of α-SMA and Ki-67, the protein ratio of LC3-II/LC3-I and Bcl-2/Bax, and the protein and mRNA expressions of Bcl-2 and Beclin-1, while upregulating the protein expressions of cleaved Caspase-3 and p-mTOR, and the protein and mRNA expressions of Bax, Caspase-3, P53, and mTOR. Bud&EEL had better effects than single-use Bud on improving airway remodeling, promoting apoptosis, and regulating the expressions of autophagy- and apoptosis-related proteins. This study suggested that the effects of coadministration of EEL and Bud on regulating apoptosis and autophagy were better than those of single-use Bud treatment, and that might be the mechanism of attenuating airway remodeling, providing an alternative therapy for asthma.

Evaluation of the effect of CaD on the bone structure and bone metabolic changes in senile osteoporosis rats based on MLP-ANN methods.

Senile osteoporosis (SOP) is a related disease of systematic degenerative changes in bones during natural aging. Increasing age is an important factor in its pathogenesis. This experiment was to evaluate the comprehensive effect of calcium with vitamin D3 (CaD) on SOP based on multilayer perception (MLP)-artificial neural network (ANN) methods. 15-month-old male Sprague-Dawley rats were administered CaD for 2 months, while 3-, 6-, 9-, 12-, 15- and 17-month-old rats were used as the mature or aging control groups. We detected the bone mass and bone mineral density (BMD), performed biomechanical testing and measured micro-CT properties to evaluate the degree of osteoporosis. Levels of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP), and the ratio of ALP to TRACP both in serum and bone were measured for the evaluation of the bone turnover rate. The bone mRNA and protein expression of ATP6v0d2, IGF-1, BMP2, M-CSF, Wnt5a and TGF-ß1 were detected by western blotting (WB), immunofluorescence (IF) and quantitative real time polymerase chain reaction (qRT-PCR) for evaluating bone metabolism in the bone microenvironment. The MLP-ANN model was constructed and used to evaluate the importance of related parameters and the comprehensive action of CaD. Our data showed that bone mass, BMD, maximal load, ultimate displacement, ALP and TRACP in serum and tibia, and the protein and mRNA expressions of ATP6v0d2, IGF-1, BMP2, M-CSF, Wnt5a and TGF-ß1 in tibia reached a peak in 6 m rats, and then were gradually decreased with the increase of age to the lowest in 17 m rats. This study demonstrated the degeneration of the bone structure and bone metabolism in SOP rats during the aging process of rats aged 3 to 17 months. CaD could effectively increase bone mass and bone strength, alleviate the degradation of the bone microstructure and rebalance bone remodeling. In addition, the MLP model was a comprehensive method for evaluating the effects of drugs on SOP, which provided a new direction for future drug and nutrition evaluation.

Combined Extracts of Herba Epimedii and Fructus Ligustri Lucidi Rebalance Bone Remodeling in Ovariectomized Rats.

This study aimed to investigate the osteoprotective effect and the possible molecular mechanisms of the combined extracts of Herba Epimedii and Fructus Ligustri Lucidi on postmenopausal osteoporosis (PMOP). Forty-eight female SD rats were sham-operated (Sham, n = 8) or ovariectomized (OVX, n = 40). Then after a week, OVX rats were divided randomly into five groups (n = 8 in each group): OVX, extracts of Herba Epimedii (HE, 0.35 g/kg), extracts of Fructus Ligustri Lucidi (FLL, 0.35 g/kg), combined extracts of HE and FLL (HE & FLL, 0.20 g/kg HE plus 0.15 g/kg FLL), and Raloxifene hydrochloride (RH, 6.25 mg/kg) groups. All groups were administered once daily for 12 weeks. Indicators related to bone remodeling were detected, including estradiol (E2), bone mineral density (BMD), maximal load, ultimate deflection, micro-CT properties, tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) levels in serum and bone, and the protein and mRNA expression of bone turnover markers (RANKL, M-CSF, Wnt5a, Atp6v0d2, OPG, IGF-1, TGF-ß1, and Bmp-2). Results showed that the combined extracts could increase serum E2 levels and BMD, enhance bone strength, reserve bone microstructure degeneration, promote bone formation, and inhibit bone resorption through upregulating the mRNA and protein expression of OPG, IGF-1, TGF-ß1, and Bmp-2, while downregulating RANKL, M-CSF, Wnt5a, and Atp6v0d2. These findings demonstrated that the combined extracts of Herba Epimedii and Fructus Ligustri Lucidi with bone protective effects on OVX rats might be an alternative medicine for the treatment of PMOP.

Effects of the combined extracts of Herba Epimedii and Fructus Ligustrilucidi on airway remodeling in the asthmatic rats with the treatment of budesonide.

BACKGROUND: Asthma is characterized by chronic airway inflammation, leading to structura1 changes in the airway, collectively termed airway remodeling. Airway remodeling is thought to contribute to airway hyper responsiveness and irreversible airflow limitation. The combination of Herba Epimedii (HE) and Fructus Ligustri Lucidi (FLL) decoction and the systemic administration of glucocorticoids (GC) had a synergistic inhibitory action on airway inflammation in the asthmatic model rats. However, the effects of the combination on airway remodeling have not been studied and compared. In the present study, we investigated the effects of the co-administration of combined extracts of HE and FLL with inhaled GC (budesonide) on airway remodeling in the rat asthmatic model induced by ovalbumin (OVA). METHODS: Male Sprague-Dawley rats were sensitized to intraperitoneal OVA followed by repetitive OVA challenge for 7 weeks. Treatments included extracts of HE and FLL (Extracts for short, 100 mg/kg by gastric perfusion), budesonide (1 mg budesonide suspension in 50 ml sterile physiological saline, 3 rats in an ultrasonic nebulizer by nebulized inhabation with a flow of 1.6 ml/min for 30 min), and co-administration of extracts of HE and FLL with budesonide (Co-administration for short) for 4 weeks. Lung histomorphometry and bronchoalveolar lavage fluid (BALF) cell count were assessed 24 h after the final OVA challenge. Levels of interleukin (IL)-4, IL-5 and IgE were measured by ELISA. Expressions of Collagen I and Collagen III were tested by immunohistology. Expressions of transforming growth factor (TGF) -ß1, TGF-ß2 and Smads mRNA were measured by quantitative real-time PCR. RESULTS: Extracts, budesonide and Co-administration significantly reduced allergen-induced increases in the serum levels of IL-4, IL-5 and IgE, the number of eosinophils in BALF, goblet cell hyperplasia, Collagen III integral optical density (IOD) and the mRNA expression of TGF-ß2 and Smad2. Extracts and Co-administration could depress the IOD level of Collagen I and the positive area of Collagen I and Collagen III. Budesonide and Co-administration significantly alleviated the thickening of airway wall. Only Co-administration significantly decreased collagen deposition according to the morphometry of Masson's-stained lung sections, the thickening of airway smooth muscle layer, the number of lymphocytes in BALF and the mRNA expression of TGF-ß1 and Smad3, and this was associated with a significant increase in levels of Smad7 mRNA. CONCLUSIONS: The findings suggested that the combination of budesonide and the herbal extracts had a better synergistic effect on airway remodeling in OVA-reduced asthma rats than the single use of budesonide.

Active Ingredients of Epimedii Folium and Ligustri Lucidi Fructus Balanced GR/HSP90 to Improve the Sensitivity of Asthmatic Rats to Budesonide.

This study aimed to investigate the possible molecular mechanisms of active ingredients of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) combined with Budesonide (Bun) in asthmatic rats. Rats were divided into 5 groups, including normal group, asthma model group, Bun group, group of active ingredients of EL and LLF (EL), and group of coadministration of Bun with EL (Bun&EL). The asthmatic model was prepared by ovalbumin sensitizing and challenging. Lymphocyte apoptosis, GR protein and binding, and the protein and mRNA of GRα, GRß, and HSP90 were tested. The results showed that Bun&EL ① markedly increased lymphocyte apoptosis, GR and HSP90 protein, and GR binding in BALF and ② enhanced the expressions of GRα and HSP90 and the ratio of GRα to GRß or to HSP90 both in protein and in mRNA levels in lung, ③ while decrease occurred in GRß mRNA and the mRNA ratio of GRß to HSP90 compared with asthma or Bun group. Moreover, there was a significant correlation between GRα and GRß in protein level, or between GRα and HSP90 both in protein and in mRNA levels. EL may effectively enhance the sensitivity of asthmatic rats to Bun via balancing GR/HSP90. And these findings will be beneficial for the treatment of asthma in the future.

Effects of the combined Herba Epimedii and Fructus Ligustri Lucidi on bone turnover and TGF-ß1/Smads pathway in GIOP rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney deficiency is the main pathogenesis of osteoporosis based on the theory of "kidney governing bones" in traditional Chinese medicine (TCM). Combined Herba Epimedii and Fructus Ligustri Lucidi, based on traditional Chinese formula Er-Zhi pills, were frequently used in TCM formulas that were prescribed for kidney tonifying and bone strengthening. However, it is unclear whether the combination of the two herbs may have a protective influence on glucocorticoid-induced osteoporosis (GIOP). The objective of this study was to evaluate the therapeutic effects and the underlying molecular mechanism of the decoction and the active fractions of the combined herbs in GIOP rats. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into seven groups, including the normal control (NC), GIOP model (MO), active fractions low (100mg/kg, LAF), active fractions high (200mg/kg, HAF), decoction low (3.5g/kg, LD), decoction high (7g/kg, HD) and Calcium with Vitamin D3 (0.2773g/kg, CaD)-treated group. The GIOP model was established by intramuscular injection of dexamethasone (1mg/kg) twice a week for 8 weeks. Different kinds of indicators were measured, including bone mineral density (BMD), bone biomechanical properties, serum bone alkaline phosphatase (b-ALP), serum bone γ-carboxyglutamic acid-containing protein (BGP), serum bone morphogenetic protein-2 (BMP-2), serum tartrate-resistant acid phosphatase (TRACP) and serum carboxy terminal cross linked telopeptide of typeⅠcollagen (ICTP), bone mineral content (BMC) and bone structured histomorphometry. The protein and mRNA expression of TGF-ß1, Smad2, Smad3, Smad4 and Smad7 were detected by Western blotting (WB) and quantitative real time polymerase chain reaction (qRT-PCR), respectively. RESULTS: Administration of combined Herba Epimedii and Fructus Ligustri Lucidi decoction and combined active fractions could significantly prevent GC-induced bone loss by increasing the contents of serum b-ALP, BGP and BMP-2 as the markers of bone formation, reducing the serum TRACP and ICTP contents to inhibit bone resorption and enhancing BMC. They could also attenuate biomechanical properties and BMD reduction, deterioration of trabecular architecture in MO rats. The mRNA and protein expressions of TGF-ß1, smad2, smad3 and smad4 were up-regulated, and the mRNA and protein expression of Smad7 was down-regulated following combined Herba Epimedii and Fructus Ligustri Lucidi treatment. CONCLUSION: Combination of Herba Epimedii and Fructus Ligustri Lucidi exhibited protective effects on promoting bone formation and precluding bone resorption. The underlying mechanism may be attributed to its regulations on TGF-ß1/Smads pathway. The substance bases of the combined herbs on anti-osteoporosis were total flavonoids of Herba Epimedii, total iridoids and flavonoids of Fructus Ligustri Lucidi.