Decoding the mechanism of Eleutheroside E in treating osteoporosis via network pharmacological analysis and molecular docking of osteoclast-related genes and gut microbiota.

Objective: Eleutheroside E (EE) is an anti-inflammatory natural compound derived from the edible medicinal herb Acanthopanax senticosus. This study aims to investigate the underlying mechanism of the anti-osteoporosis action of EE through network pharmacology, molecular docking and gut microbiota. Materials and methods: Network pharmacology was used to explore the potential core targets and main pathways mediated by EE in osteoporosis (OP) treatment. Molecular docking was exploited to investigate the interactions between the active anti-OP compounds in EE and the potential downstream targets. Following the multi-approach bioinformatics analysis, ovariectomy (OVX) model was also established to investigate the in vivo anti-OP effects of EE. Results: The top 10 core targets in PPI network were TP53, AKT1, JUN, CTNNB1, STAT3, HIF1A, EP300, CREB1, IL1B and ESR1. Molecular docking results that the binding energy of target proteins and the active compounds was approximately between -5.0 and -7.0 kcal/mol, which EE has the lowest docking binding energy with HIF1A. Enrichment analysis of GO and KEGG pathways of target proteins indicated that EE treatment could potentially alter numerous biological processes and cellular pathways. In vivo experiments demonstrated the protective effect of EE treatment against accelerated bone loss, where reduced serum levels of TRAP, CTX, TNF-α, LPS, and IL-6 and increased bone volume and serum levels of P1NP were observed in EE-treated mice. In addition, changes in gut microbiota were spotted by 16S rRNA gene sequencing, showing that EE treatment increased the relative abundance of Lactobacillus and decreased the relative abundance of Clostridiaceae. Conclusion: In summary, these findings suggested that the characteristics of multi-target and multi-pathway of EE against OP. In vivo, EE prevents the onset of OP by regulating gut microbiota and inflammatory response and is therefore a potential OP drug.

Danshen (Salvia miltiorrhiza) injection suppresses kidney injury induced by iron overload in mice.

OBJECTIVES: Excessive iron can accumulate in the kidney and induce tissue damage. Danshen (Salvia miltiorrhiza) injection is a traditional Chinese medicinal preparation used for preventing and treating chronic renal failure. The aim of the present study was to evaluate the effects of treatment with Danshen injection on iron overload-induced kidney damage. METHODS: Mice were mock-treated with saline (control group) or given a single dose of iron dextran without treatment (iron overload group, 50 mg/kg/day for 2 weeks) or with daily treatments of low-dose Danshen (3 g/kg/day), high-dose Danshen (6 g/kg/day) or deferoxamine (100 mg/kg/day). RESULTS: Treatment of iron-overloaded mice with Danshen injection led to significant improvements of body weight and decreased iron levels in the kidney. Danshen injection treatment also reduced concentrations of blood urea nitrogen, creatinine and malondialdehyde and enhanced glutathione peroxidase and superoxide dismutase activities. Histopathological examinations showed that Danshen injection ameliorated pathological changes and reduced iron deposition in kidneys of iron overloaded mice. Furthermore, the treatment was demonstrated to suppress apoptosis in nephrocytes. CONCLUSIONS: These results indicated that Danshen injection exerted significant renal protective effects in iron-overloaded mice, which were closely associated with the decrease of iron deposition and suppression of lipid peroxidation and apoptosis in the kidney.

Salvia miltiorrhiza (Danshen) injection ameliorates iron overload-induced cardiac damage in mice.

The traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), first recorded in the "Shen Nong's Herbal Classic", has long been used to treat cardiovascular conditions, although the mechanism(s) underlying its effects remain unclear. Here, an iron dextran injection (50 mg · kg⁻¹ per day) was delivered intraperitoneally to establish a mouse model for investigating the ameliorative effects of Danshen injection (low dose at 3 g · kg⁻¹ per day or high dose at 6 g · kg⁻¹ per day) on iron overload-induced cardiac damage. The iron-chelating agent deferoxamine (100 mg · kg⁻¹ per day) was administered as a positive control. The main constituents of Danshen injection, salvianic acid A (danshensu), protocatechuic aldehyde, and salvianolic acid B, were quantified at concentrations of 2.15, 0.44, and 1.01 mg · mL⁻¹, respectively, using HPLC with UV detection. Danshen injection significantly lowered cardiac iron deposition and the concentration of the lipid oxidation product malondialdehyde, as well as improved cardiac superoxide dismutase and glutathione peroxidase levels in iron-overloaded mice. Serum levels of creatine kinase, creatine kinase isoenzyme, and lactate dehydrogenase in the iron-overloaded mice were significantly elevated (up to ~ 160 %), whereas their activities were downregulated by Danshen injection by 25 ~ 35 % at the high dose and by ~ 20 % at the low dose. Morphological changes of cardiac tissue analyzed by hematoxylin and eosin staining indicated that lesions induced by iron overload could be ameliorated by Danshen injection dose-dependently. Altogether, these results illustrated that the protective effects of Danshen injection were at least in part due to decreased iron deposition and inhibition of lipid peroxidation.

Multitargeted inhibition of hepatic fibrosis in chronic iron-overloaded mice by Salvia miltiorrhiza.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza (SM, also known as Danshen) is a well-known Chinese medicinal herb, which has shown hepatoprotective effects with anti-fibrotic, anti-oxidative, anti-inflammatory and anti-apoptotic properties. To explore the effects and potential mechanism of SM against hepatic fibrosis induced by chronic iron overload in mice. MATERIALS AND METHODS: Sixty male mice were randomized into five groups (n=12 in each group): control (saline), iron overload, iron overload with low-dose SM (3g/kg/day), iron overload with high-dose SM (6g/kg/day) and iron overload with deferoxamine (100mg/kg/day) groups. The iron overload model was established by intraperitoneal injection with iron dextran at 50mg/kg body weight/day, and the entire course lasted for 7 weeks. The major constituents of SM injection were quantified by high performance liquid chromatography. Changes of hepatic iron, hydroxyproline (Hyp), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were assayed by standard procedures. Protein expression levels of type I collagen, type III collagen, tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α) were analyzed by immunohistochemistry, and mRNA levels of transforming growth factor-ß (TGF-ß), matrix metal proteinase-9 (MMP-9) and caspase-3 were detected by RT-PCR. Morphological changes were observed with Prussian blue, Masson's trichrome and hematoxylin-eosin staining. RESULTS: Treatment of chronic iron-overloaded mice with SM dose-dependently ameliorated changes in hepatic morphology and coefficient, reduced iron deposition and Hyp content, suppressed overexpression of type I collagen and type III collagen, downregulated expression of TGF-ß mRNA, and upregulated expression of MMP-9 mRNA in the liver. Moreover, SM treatment contributed to decreased MDA content, increased SOD activity and GSH content, while it reduced expression of TNF-α, IL-1α and caspase-3. CONCLUSIONS: SM displayed anti-fibrotic activity in the liver induced by chronic iron overload, which may be attributed to multitargeted inhibition of iron deposition and collagen accumulation, as well as oxidative stress, inflammation and apoptosis.

Nephroprotective effect of calcium channel blockers against toxicity of lead exposure in mice.

Exposure to lead (Pb) can induce kidney damage, which is related to induction of oxidative damage and disturbance of intracellular calcium homeostasis. Pb can readily permeate through dihydropyridine-sensitive L-type calcium channels and accumulate within cells. The objective of this study was to investigate protective effects of calcium channel blockers (CCBs) verapamil and nimodipine on nephrotoxicity induced by Pb acetate in mice. One hundred and twenty male mice were randomly divided into 6 groups: control, Pb, low-dose verapamil, high-dose verapamil, low-dose nimodipine and high-dose nimodipine (n=20 per group). Pb acetate was injected intraperitoneally (i.p.) at 40 mg/kg body weight/day for 10 days to establish the Pb toxicity model. While control mice received saline, mice of the treated groups simultaneously received i.p. injections of verapamil or nimodipine daily for 10 days. Both verapamil and nimodipine showed protection against Pb-induced kidney injury, including alleviation of renal pathological damage and decreasing the level of Pb in kidney homogenate and extent of apoptosis in nephrocytes. Moreover, verapamil and nimodipine significantly down-regulated levels of blood urea nitrogen and creatinine in the serum. In addition, verapamil and nimodipine administration decreased malondialdehyde content and increased activities of super oxide dismutase activity and glutathione peroxidase in the kidney homogenate. The findings in the present study implicate the therapeutic potential of CCBs for Pb-induced nephrotoxicity, which were at least partly due to the decrease of Pb uptake and inhibition of lipid peroxidation.

Mechanism of protective effects of Danshen against iron overload-induced injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen (Salvia miltiorrhiza) has been widely prescribed in traditional folk medicine for treatment of hepatic and cardiovascular diseases in China and other Asian countries for several hundred years. MATERIALS AND METHODS: Sixty male mice were randomly divided into five groups: control, iron overload, low-dose Danshen (L-Danshen, 3g/kg/day), high-dose Danshen (H-Danshen, 6g/kg/day) and deferoxamine (DFO) groups (n=12 per group). Iron dextran was injected intraperitoneally (i.p.) at 50mg/kg body weight/day to establish the iron overload model. While control mice received saline, mice of the treated groups simultaneously received (i.p.) injections of L-Danshen, H-Danshen or DFO daily for 2 weeks. At the end of the experiment, changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST), glutathione peroxidase (GSH-Px), superoxide desmutase (SOD) and malondialdehyde (MDA) were measured, and histological changes were observed by Prussian blue or hematoxylin and eosin staining of the liver. Apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling. RESULTS: Treatment of iron overloaded mice with either low or high doses of Danshen not only significantly attenuated the hepatic dysfunction (ALT/AST levels), decreased the content of MDA and increased the activities of GSH-Px and SOD, it also suppressed apoptosis in hepatocytes. Histopathological examination showed that treatment with Danshen reduced iron deposition and ameliorated pathological changes in the liver of iron overloaded mice. CONCLUSIONS: Danshen demonstrated significant protective effects in the liver of iron overloaded mice, which were at least partly due to the decrease of iron deposition and inhibition of lipid peroxidation and hepatocyte apoptosis.