Xueshuantong injection combined with Salvianolate lyophilized injection improves the synaptic plasticity against focal cerebral ischemia/reperfusion injury in rats through PI3K/AKT/mTOR and RhoA/ROCK pathways.

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. Salvianolate lyophilized injection (SLI) made from the aqueous extraction of salvia miltiorrhiza and Xueshuantong injection (lyophilized) (XST) made from the Panax Notoginseng extraction are two herbal standardized preparations that have been widely used in China for the treatment of ischemic stroke. In this study, we investigated the neuroprotective effects of XST combined with SLI in the recovery stage of middle cerebral artery occlusion / reperfusion (MCAO/R) injury rat. Wistar rats were subjects to MCAO/R, then were treated with SLI or XST alone, or with their combination (1X1S) via tail injection daily for 14 days. The pathological status of the brain was detected by neurological deficit scores, TTC, regional cerebral blood flow and Nissl staining. Golgi-Cox staining was used to assess dendritic, axonal and synaptic remodeling. The expression of MAP-2, ß-Tubulin, PSD95, SYN, BDNF and VEGF were analyzed by western blotting and immunofluorescence. The results showed that administration of 1X1S not only significantly decreased neurological scores and infarct volumes, but also increased regional cerebral blood flow, strengthened dendritic and synaptic remodeling compared with XST, SLI used alone. And the mechanism of combined of 1X1S to exert neuroprotection may be associated with PI3K/ AKT/ mTOR and RhoA/ROCK2 pathways. Overall, these findings suggest that combination of XST and SLI promotes dendritic spine density and synaptic plasticity via upregulation of the PI3K/ AKT/ mTOR pathways and inhabitation the RhoA/ROCK2 signaling pathway in rat with MCAO/R, showing its multiple-action-multiple-target efficacy and suggest a potential new strategy for ischemia.

Anti-perimenopausal osteoporosis effects of Erzhi formula via regulation of bone resorption through osteoclast differentiation: A network pharmacology-integrated experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi formula (EZF) consists of Ecliptae herba (EH) and Fructus Ligustri Lucidi (FLL) at a ratio 1:1, and constitutes a well-known formula in China that is commonly used for treating menopausal diseases. AIM OF THE STUDY: In this study, we explored the pharmacologic actions and potential molecular mechanisms underlying EZF's action in preventing and treating osteoporosis. MATERIALS AND METHODS: The active components and related targets of EZF's anti-osteoporotic effects were predicted by network pharmacology, and functional enrichment analysis was also performed. We then used an osteoporosis model of ovariectomized (OVX) mice to detect the effects of EZF on osteoporosis. RESULTS: The results from network pharmacology identified a total of 10 active ingredients from EH and 13 active ingredients from FLL that might affect 65 potential therapeutic targets. GO enrichment analysis revealed that EZF affected bone tissue primarily via hormone (particularly estradiol)-related pathways and bone resorption by osteoclast differentiation. KEGG analysis demonstrated that bone-related factors such as Runt-related transcription factor 2 (Runx2), Ca2, estrogen receptor1 (ESR1), androgen receptors (AR), and TNFα served as the primary targets during osteoclastic differentiation. In vivo experiments showed that the formula significantly improved the diminution in estrogen and the subsequent uterine atrophy induced by ovariectomy (P < 0.01 or 0.05), implying that the EZF exerted its actions via regulation of estradiol and the nourishing effects of the uterus in OVX mice. Dual-energy X-ray absorptiometry and micro-CT showed that EZF significantly inhibited bone loss and improved bone micro-architecture by statistically increasing the number of bone trabeculae and decreasing the separation of bone trabeculae in OVX mice (P < 0.01 or 0.05); EZF also inhibited bone loss and enhanced bone-fracture load. Furthermore, we confirmed that EZF reduced the calcium concentrations, augmented protein and mRNA levels for Runx2 in the bone marrow, and reduced PPARγ levels. RANKL-a key downstream regulatory protein of many targets that was referred to in our results of network pharmacology as being involved in the regulation of osteoclastogenesis-was significantly diminished by EZF; it also elevated OPG content. In addition, we used monocytes of bone-marrow origin to detect the effects of the potential components of EZF on osteoclast differentiation and found that wedelolactone, oleanolic acid, echinocystic acid, luteolin, and luteolin-7-o-glucoside significantly inhibited osteoclast differentiation from monocytes induced by 25 ng/mL MCSF and 50 ng/mL RANKL (P < 0.01 or 0.05). CONCLUSIONS: Our present study indicated that EZF significantly inhibited the bone loss induced by OVX in mice by its regulation of estradiol combined with the nourishing effect of the uterus, and that it also attenuated bone resorption by decreasing the RANKL/OPG ratio so as to inhibit osteoclast maturation.

Effects of salvianolate lyophilized injection combined with Xueshuantong injection in regulation of BBB function in a co-culture model of endothelial cells and pericytes.

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. The salvianolate lyophilized injection (SLI) and Xueshuantong Injection (XST) are two standardized Chinese medicine injections which have been widely used in the treatment of cerebrovascular diseases. Salvianolic acid B (Sal B) and Notoginsenoside R1 (NR1) is respectively one of the active constituents of SLI and XST, which have certain effects on stroke. In this study, we established a co-culture of endothelial cells and pericytes for oxygen-glucose deprivation/reperfusion (OGD/R) injury model to study the effects of SLI and Sal B or XST and NR1 alone, or with their combinations (1S1X) in regulation of BBB function. The results showed that compared with the OGD/R group, treatment with SLI, XST and SalB and NR1 can significantly increase the TEER, reduce the permeability of Na-Flu, enhance the expression of tight junctions (TJs) between cells, and stabilize the basement membrane (BM) composition. In addition, the combination of 1S1X is superior to the XST or SLI alone in enhancing the TJs between cells and stabilizing the BM. And the active components SalB and NR1 can play a strong role in these two aspects, even with the whole effects. Furthermore, the study showed that XST, Sal B and NR1 increases in Ang-1and Tie2, while decrease in Ang-2 and VEGF protein expressions. Overall, these findings suggest that SLI combined with XST (1X1S) has protective effects on co-culture of endothelial cells and pericytes after OGD/R. Moreover, its protective effect might be associated with increase of TJs and BMs through activation of Ang/Tie-2 system signaling pathway.

Hepatoprotective Effect of Cereal Vinegar Sediment in Acute Liver Injury Mice and Its Influence on Gut Microbiota.

Cereal vinegar sediment (CVS) is a natural precipitate formed during the aging process of traditional grain vinegar. It has been used as Chinese traditional medicine, while its composition and function are reported minimally. In this study, we measured CVS in terms of saccharide, protein, fat and water content, and polyphenol and flavonoid content. Furthermore, we determined the amino acids, organic acids, and other soluble metabolites in CVS using reverse-phase high-performance liquid chromatography (RP-HPLC), HPLC, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) platforms. The hepatoprotective effect of CVS was evaluated in acute CCl4-induced liver injury mice. Administration of CVS for 7 days prior to the CCl4 treatment can significantly decrease liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and reactive oxygen species (ROS) levels, compared with those in the hepatic injury model group. The gut microbiota was changed by CCl4 administration and was partly shifted by the pretreatment of CVS, particularly the Muribaculaceae family, which was increased in CVS-treated groups compared with that in the CCl4 administration group. Moreover, the abundances of Alistipes genus and Muribaculaceae family were correlated with the liver ALT, AST, and malondialdehyde (MDA) levels. Our results illustrated the composition of CVS and its hepatoprotective effect in mice, suggested that CVS could be developed as functional food to prevent acute liver injury.

Xueshuantong for Injection (Lyophilized, ) Alleviates Streptozotocin-Induced Diabetic Retinopathy in Rats.

OBJECTIVE: To investigate the ameliorate effect and underlying mechanism of Xueshuantong for Injection (Lyophilized, , XST) in streptozocin (STZ)-induced diabetic retinopathy (DR) rats. METHODS: Diabetes mellitus (DM) model was induced by intraperitoneal (i.p.) injection of STZ (60 mg/kg) in Sprague-Dawley rats. Diabetic rats were randomized into 3 groups (n=10) according to a random number table, including DM, XST50 and XST100 groups. XST treatment groups were daily i.p. injected with 50 or 100 mg/kg XST for 60 days, respectively. The control and DM groups were given i.p. injection with saline. Blood glucose level and body weight were recorded every week. Histological changes in the retina tissues were observed with hematoxylin-eosin staining. Apoptosis and inflammation related factors, including cleaved caspase-3, glial fifibrillary acidic protein (GFAP), tumor necrosis factor-α (TNF-α) and intercellular cell adhesion molecule-1 (ICAM-1) were detected by Western blot or real-time polymerase chain reaction. Then, the levels of advanced glycation end product (AGE) and its receptor (RAGE) were investigated. Tight junctions proteins (Zonula occludens-1 (ZO-1), Occludin and Claudin-5) of blood-retinal barrier were detected by Western blot. The levels of retinal fifibrosis, transforming growth factor-ß1 (TGF-ß1)-Smad2/3 signaling pathway were evaluated at last. RESULTS: There was no signifificant difference in the body weight and blood glucose level between XST and DM groups (P>0.05). Compared with the DM group, XST treatment signifificantly increased the retinal thickness of rats (P<0.05 or P<0.01), and suppressed cleaved caspase-3 expression (P<0.01). XST increased the protein expressions of ZO-1, Occludin and Claudin-5 and decreased the mRNA expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 (P<0.05 or P<0.01). Moreover, XST signifificantly reduced the productions of AGE and RAGE proteins in the retina of rats (P<0.05 or P<0.01), suppressed the over-expression of TNF-α, and decreased the elevated level of ICAM-1 in retina of rats (P<0.05 or P<0.01). XST signifificantly reduced the levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), TGF-ß1 and phosphorylation of Smad2/3 protein in rats (P<0.05 or P<0.01). CONCLUSIONS: XST had protective effects on DR with possible mechanisms of inhibiting the inflammation and apoptosis, up-regulating the expression of tight junction proteins, suppressing the productions of AGE and RAGE proteins, and blocking the TGF-ß/Smad2/3 signaling pathway. XST treatment might play a role for the future therapeutic strategy against DR.

Molecular Pathway of Psoralidin-Induced Apoptosis in HepG2 Cell Line.

OBJECTIVE: To test the role of psoralidin in human liver cancer HepG2 cells in vitro. METHODS: Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexin V then sorted by flow cytometry. Protein expressions of caspase-3, caspase-8, caspase-9, Bax, Bid, Bcl-2, Bcl-xL and p53 were examined by western blot while activity of caspase-3, -8 and -9 were also determined. RESULTS: Psoralidin reduces cell viability greatly in a time dependent manner (64%, 40%, 21%, 12% at 2, 6, 24 and 48 h treatment with 64 µmol/L psoralidin respectively) and up-regulates activities of caspase-3, -8 and -9 in a concentration dependent manner (between 4 to 64 µmol/L). Psoralidin also increases the expression of pro-apoptosis genes Bax, Bid and p53 while decreases the expression of pro-survival genes Bcl-2 and Bcl-xL, both in a concentration dependent manner between 4 and 64 µmol/L (P<0.05 at 16 and 64 µmol/L). Caspase-3 inhibitor (Ac-DEVD-CHO at concentrations between 10 to 20 µmol/L), p53 inhibitor (pifithrin-α at 5 µmol/L) and cyclosporin A can attenuate the apoptotic effect of psoralidin. CONCLUSION: The cytotoxic role of psoralidin might work through both intrinsic and extrinsic apoptotic pathway.

The Antiosteoporosis Effects of Zhuanggu Guanjie Pill In Vitro and In Vivo.

We investigated the beneficial effects and underlying mechanisms of Zhuanggu Guanjie (ZGGJ) pill in osteoporosis in vitro and in vivo. Bone marrow macrophages from 4-6-week-old mice were cultured in the presence of macrophage colony-stimulating factor (15 ng/mL) and receptor activator of nuclear factor-κB ligand (30 ng/mL). Osteoclast differentiation was determined by quantification of tartrate-resistant acid phosphatase activity. Gelatin zymography was used to detect the activity of matrix metalloproteinases in osteoclasts. Ovariectomized rats were administered orally with estradiol valerate or ZGGJ for 8 weeks. Blood was collected to measure serum indices. Tibiae were harvested to carry out bone microcomputed tomography scanning, histomorphological analysis, and bone strength determination. ZGGJ inhibited tartrate-resistant acid phosphatase activity, matrix metalloproteinase 9 expression, and bone resorption in vitro. At doses of 0.55, 1.1, and 2.2 g/kg, ZGGJ exerted significant osteoprotective effects including inhibition of bone turnover markers and improved tibia bone strength in ovariectomized rats. Microcomputed tomographic analysis showed that ZGGJ improved the trabecular architecture with increased connectivity density and trabecular thickness and decreased trabecular spacing. These results revealed that ZGGJ prevents bone loss induced by ovariectomy in rats and that inhibition of bone resorption is involved in the bone-protective effects of ZGGJ.

Xueshuantong injection (lyophilized) combined with salvianolate lyophilized injection protects against focal cerebral ischemia/reperfusion injury in rats through attenuation of oxidative stress.

Salvianolate lyophilized injection (SLI) and Xueshuantong injection (lyophilized) (XST) are two herbal standardized preparations that have been widely used in China for the treatment of acute cerebral infarction. In this study, we investigated the neuroprotective effects of SLI combined with XST in a rat model of middle cerebral artery occlusion-reperfusion (MCAO/R). Wistar rats were subjected to 1.5 h of MCAO followed by reperfusion for 3 h, then were treated with SLI or XST alone, or with their combinations via tail vein injection daily for 3 d. Edaravone (EDI, 6 mg·kg-1·d-1) was used as a positive control drug, We showed that administration of a combination of 1X1S (XST 100 mg·kg-1·d-1 plus SLI 21 mg·kg-1·d-1) more effectively protected the ischemic brains than SLI or XST used alone. Administration of 1X1S not only significantly decreased neurological deficit scores and infarct volumes and increased regional cerebral blood flow, but also inhibited the activation of both microglia and astrocytes in the hippocampus. Furthermore, administration of 1X1S significantly decreased the levels of MDA and ROS with concomitant increases in the levels of antioxidant activity (SOD, CAT and GSH) in the brain tissues as compared with SLI and XST used alone. Moreover, administration of 1X1S remarkably upregulated the expression of Nrf-2, HO-1 and NQO-1, and downregulated the expression of Keap1 and facilitated the nuclear translocation of Nrf-2 in the brain tissues as compared with XST used alone. Our study demonstrates that a combination of 1X1S effectively protects MCAO/R injury via suppressing oxidative stress and the Nrf-2/Keap1 pathway.