Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat.

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

Six New Constituents from the Fruit of Hypericum patulum and Their Anti-Inflammatory Activity.

Four new xanthone glucosides, 3-hydroxy-2-methoxyxanthone-4-O-ß-D-glucopyranoside (1), 4,8-dihydroxy-2-methoxyxanthone-3-O-ß-D-glucopyranoside (2), 2-methoxyxanthone-5-O-ß-D-glucopyranoside (3), 4-hydroxy-2-methoxyxanthone-3-O-ß-D-glucopyranoside (4), a new phenolic acid, 4,4'-dihydroxy-3,3'-imino-di-benzoic acid monomethyl ester (5), and a new isoquinoline, methyl 6-hydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (6) were isolated from the fruit of Hypericum patulum. The structural elucidation of the isolated compounds was primarily based on HR-ESI-MS, UV, IR, 1D and 2D NMR. All compounds were evaluated for their inhibitory effect against LPS-induced NO production in RAW 264.7 cells. Compound 2, 3 exhibited moderate inhibitory activity against NO production.

[Effects of Gukang Capsules on activity and protein expression of hepatic cytochrome P450 enzymes in rats].

Gukang Capsules are often used in combination with drugs to treat fractures, osteoarthritis, and osteoporosis. Cytochrome P450(CYP450) mainly exists in the liver and participates in the oxidative metabolism of a variety of endogenous and exogenous substances and serves as an important cause of drug-metabolic interactions and adverse reactions. Therefore, it is of great significance to study the effect of Gukang Capsules on the activity and expression of CYP450 for increasing its clinical rational medication and improving the safety of drug combination. In this study, the Cocktail probe method was used to detect the changes in the activities of CYP1A2, CYP3A2, CYP2C11, CYP2C19, CYP2D4, and CYP2E1 in rat liver after treatment with high-, medium-and low-dose Gukang Capsules. The rat liver microsomes were extracted by the calcium chloride method, and protein expression of the above six CYP isoform enzymes was detected by Western blot. The results showed that the low-dose Gukang Capsules could induce CYP3A2 and CYP2D4 in rats, medium-dose Gukang Capsules had no effect on them, and high-dose Gukang Capsules could inhibit them in rats. The high-dose Gukang Capsules did not affect CYP2C11 in rats, but low-and medium-dose Gukang Capsules could induce CYP2C11 in rats. Gukang Capsules could inhibit CYP2C19 in rats and induce CYP1A2 in a dose-independent manner, but did not affect CYP2E1. If Gukang Capsules were co-administered with CYP1A2, CYP2C19, CYP3A2, CYP2C11, and CYP2D4 substrates, the dose should be adjusted to avoid drug interactions.

[Effects and correlation of ligustrazine hydrochloride-Salviae Miltiorrhizae Radix et Rhizoma compatibility on pharmacokinetics and CYP450 enzyme].

The present study investigated the effects of ligustrazine hydrochloride(LH)-Salviae Miltiorrhizae Radix et Rhizoma(SM) before and after compatibility on the pharmacokinetics of acute myocardial ischemia(AMI) rats and revealed the mechanism of pharmacokinetic changes from the perspective of metabolic enzymes. AMI rats underwent single injection of SM Glucose Injection, LH Glucose Injection, and LH-SM Glucose Injection in the caudal vein, respectively(3.78 mg·kg~(-1) salvianic acid, 0.049 mg·kg~(-1) rosmarinic acid, and 13.68 mg·kg~(-1) ligustrazine). Blood samples were collected from the orbital venous plexus at different time points, and the liver of the rats was removed after the last blood sampling. The plasma concentrations of salvianic acid, rosmarinic acid, and ligustrazine were detected by UPLC-MS/MS. Western blot was used to detect the protein expression of CYP1 A2, CYP2 C11, CYP2 C19, CYP2 D4, CYP2 E1, and CYP3 A2 in the liver of rats in each group. As revealed by the pharmacokinetic results, compared with the LH Glucose Injection group, the LH-SM Glucose Injection group showed a downward trend of T_(1/2) of ligustrazine in AMI rats and decreased AUC(P<0.05). Compared with the SM Glucose Injection, there were no significant differences in the pharmacokinetic parameters of salvianic acid and rosmarinic acid in the LH-SM Glucose Injection group. Protein expression results showed that the expression levels of CYP1 A2, CYP2 C11, CYP2 D4, CYP2 E1, and CYP3 A2 in the LH-SM Glucose Injection group increased(P<0.05) and the expression level of CYP2 C19 decreased(P<0.05) compared with those in the LH Glucose Injection group. CYP1 A2, CYP2 C11, and CYP3 A2 are isoenzymes involved in ligustrazine Ⅰ metabolism. When LH and SM were used in combination, the expression of these three enzymes increased, which changed the pharmacokinetic process in rats and accelerated the metabolism of ligustrazine.

Shenxiong glucose injection inhibits oxidative stress and apoptosis to ameliorate isoproterenol-induced myocardial ischemia in rats and improve the function of HUVECs exposed to CoCl2.

Background: Shenxiong Glucose Injection (SGI) is a traditional Chinese medicine formula composed of ligustrazine hydrochloride and Danshen (Radix et rhizoma Salviae miltiorrhizae; Salvia miltiorrhiza Bunge, Lamiaceae). Our previous studies and others have shown that SGI has excellent therapeutic effects on myocardial ischemia (MI). However, the potential mechanisms of action have yet to be elucidated. This study aimed to explore the molecular mechanism of SGI in MI treatment. Methods: Sprague-Dawley rats were treated with isoproterenol (ISO) to establish the MI model. Electrocardiograms, hemodynamic parameters, echocardiograms, reactive oxygen species (ROS) levels, and serum concentrations of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were analyzed to explore the protective effect of SGI on MI. In addition, a model of oxidative damage and apoptosis in human umbilical vein endothelial cells (HUVECs) was established using CoCl2. Cell viability, Ca2+ concentration, mitochondrial membrane potential (MMP), apoptosis, intracellular ROS, and cell cycle parameters were detected in the HUVEC model. The expression of apoptosis-related proteins (Bcl-2, Caspase-3, PARP, cytoplasmic and mitochondrial Cyt-c and Bax, and p-ERK1/2) was determined by western blotting, and the expression of cleaved caspase-3 was analyzed by immunofluorescence. Results: SGI significantly reduced ROS production and serum concentrations of cTnI and cTnT, reversed ST-segment elevation, and attenuated the deterioration of left ventricular function in ISO-induced MI rats. In vitro, SGI treatment significantly inhibited intracellular ROS overexpression, Ca2+ influx, MMP disruption, and G2/M arrest in the cell cycle. Additionally, SGI treatment markedly upregulated the expression of anti-apoptotic protein Bcl-2 and downregulated the expression of pro-apoptotic proteins p-ERK1/2, mitochondrial Bax, cytoplasmic Cyt-c, cleaved caspase-3, and PARP. Conclusion: SGI could improve MI by inhibiting the oxidative stress and apoptosis signaling pathways. These findings provide evidence to explain the pharmacological action and underlying molecular mechanisms of SGI in the treatment of MI.

Shenxiong glucose injection inhibits H2O2-induced H9c2 cell apoptosis by activating the ERK signaling pathway.

BACKGROUND: Shenxiong glucose injection (SGI) is a traditional Chinese medicine injection composed of water extract of Salvia miltiorrhiza and Ligustrazine hydrochloride. SGI has shown strong antioxidant and anti-apoptotic properties. However, the mechanisms underlying its anti-apoptotic effect need to be addressed. METHODS: H9c2 cell apoptosis model was established by treatment of hydrogen peroxide (H2O2). Cell survival rates were examined by MTS assay, cell apoptosis rates were determined by flow cytometry, levels of intracellular ROS were assessed by ROS kit, proteome phosphorylation was determined by phosphoproteomic analysis, and extracellular signal-regulated kinase (ERK), phosphorylated ERK, phosphorylated c-Jun, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, and cleaved caspase-3 were examined by Western blot. RESULT: SGI showed protective effects against H2O2-induced reduced cell viability, elevated ROS, and increased apoptosis in H9c2 cells. Phosphorylation proteomics detected a total of 3369 proteins with 78 protein of upregulated phosphorylation and 104 protein of downregulated phosphorylation. Kyoto Encyclopedia Genes and Genomes pathway analyses of differentially phosphorylated proteins showed that the ERK pathway, the downstream pathway of the focal adhesion pathway related to apoptosis, was highly enriched, and the phosphorylation levels of ERK and c-Jun were confirmed by Western blot. In addition, the ERK pathway inhibitor PD98059 significantly inhibited the anti-apoptotic effect of SGI. CONCLUSION: SGI antagonizes H2O2-induced cell apoptosis by activating the ERK pathway.

[Shenxiong Glucose Injection inhibits H_2O_2-induced apoptosis of H9c2 cells by activating PI3K/AKT pathway].

The aim of this paper was to explore the mechanism of Shenxiong Glucose Injection antagonizing apoptosis of H9 c2 cells induced by H_2O_2. H9 c2 cells were pretreated with 1. 7%,3. 4% and 6. 8% Shenxiong Glucose Injection,and then H_2O_2 was introduced to induce apoptosis in vitro. Cell viability was detected by MTS assay,morphological changes of apoptosis were observed by AO/EB fluorescence staining,apoptosis rate was detected by Annexin/PI method,cell expression profile was detected by gene chip technology,the mRNA of PIK3 CA,Bcl-2,Bax,caspase-3 and GAPDH were detected by qRT-PCR,the protein expression levels of PIK3 CA,AKT,P-AKT,Bcl-2,Bax and caspase-3 were detected by Western blot,and the contents of LDH and MDA were detected by kit. The results showed that Shenxiong Glucose Injection of different concentrations significantly increased the viability of H9 c2 cells treated with H_2O_2( P<0. 01),and reversed H_2O_2-induced apoptosis( P< 0. 01). The microarray experiments showed that 138 genes were altered in H9 c2 cells after treatment with Shenxiong Glucose Injection. The differential expression fold of PIK3 CA associated with PI3 K/AKT pathway was 3. 59. The results of qRT-PCR and Western blot showed that Shenxiong Glucose Injection could down-regulate the mRNA and protein expression levels of caspase-3( P<0. 01),up-regulate the mRNA and protein expression level of PIK3 CA and Bcl-2( P<0. 01),and up-regulate the phosphorylation levels of AKT( P<0. 01) in H_2O_2-treated H9 c2 cells. The protective effect of Shenxiong Glucose Injection on H_2O_2 cells injury was significantly inhibited by LY294002,a PI3 K/AKT pathway inhibitor. The results suggested that Shenxiong Glucose Injection may inhibit H_2O_2-induced H9 c2 cells apoptosis by regulating PI3 K/AKT signaling pathway.

[Anti-oxidation and anti-apoptosis mechanism of Xinshao formula on cerebral ischemia reperfusion injury].

The cerebral ischemia-reperfusion model was established to investigate the anti-oxidation and anti-apoptosis mechanism of Xinshao formula on the cerebral ischemia reperfusion injury in rats. SD rats were randomly divided into five groups: sham operation group, model group, and low, middle and high-dose Xinshao formula groups (0.31, 0.62, 1.25 g·kg⁻¹). After administration with Xinshao formula for 7 days, the rats were used to establish the cerebral ischemia-reperfusion model. The neurological behavior was evaluated. TTC staining was implemented to determine the volume of cerebral infarction. The levels of ROS, SOD, GSH-PX, NO and iNOS in serum were examined, and the mRNA and protein levels of Bcl-2, Bax and caspase 3 in hippocampal CA1 were detected by qRT-PCR, immunohistochemical assay and Western blot. It is found that Xinshao formula could significantly reduce the symptoms of nervous function and the volume of cerebral infarction in MACO rats. Compared with model group, the rats in Xinshao formula group showed increases in the activities of SOD and GSH-PX (P<0.01), and decreases in the activities of iNOS and the contents of NO, ROS and MDA (P<0.01). In addition, Xinshao formula could down-regulate the mRNA and protein levels of Bax and caspase 3 (P<0.01), and up-regulated those of Bcl-2 (P<0.01) in MACO rats. In conclusion, Xinshao formula showed aprotective effect on cerebral ischemia reperfusion injuryin rats, which may be associated with the promotion of anti-oxidation and anti-apoptosis.

[Protective effect of Polygonum orientale flower extract on H2O2-induced oxidative damage of HUVEC cells].

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H2O2-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H2O2 was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 µmol·L⁻¹ H2O2 for 0.5 h. Treatment of H2O2 also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H2O2 model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H2O2-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.