Effect and mechanism of Dahuang Zhechong Pills in improving liver aging in rats by regulating ROS-mediated PI3K/Akt/FoxO4 signaling pathway / 中国中药杂志

Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.

Effects and mechanisms of total flavones of Abelmoschus manihot in attenuating diabetic tubulopathy by targeting endoplasmic reticulum stress-induced cell apoptosis / 中国中药杂志

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.

Physical and chemical properties of pulp capping materials based on bioactive glass / 北京大学学报(医学版)

OBJECTIVE@#To investigate the physical and chemical properties of pulp capping materials based on bioactive glass (BG).@*METHODS@#Novel BG pulp capping materials were composed of powder and fluid. The powder was BG (82.36% SiO2, 15.36% CaO, and 2.28% P2O5) synthesized by using the sol-gel method combined with template technology. Two kinds of fluid were provided: (1) phosphate buffer (PB) solution and (2) phosphate buffer solution with 1% sodium alginate (SA) addition. After mixing the powder and fluid, BG-PB and BG-PB-SA were prepared. Setting time and compressive strength of the BG pulp capping materials were tested by setting time loading system and mechanical testing machine. Statistical analysis was performed using the independent sample t-test, with the significance set at 0.05. pH meters was used to test the pH of the BG pulp capping materials and mineral trioxide aggregate (MTA). The sealing ability of the BG pulp capping materials and MTA was tested by methylene blue dye leakage model. Statistical analysis was performed using one-way ANOVA analysis and LSD multiple comparison, with the significance set at 0.05.@*RESULTS@#(1) Setting time: the initial and final setting time of BG-PB were (7.2±0.3) min and (12.7±0.9) min, respectively. And the initial and final setting time of BG-PB-SA was (7.5±0.3) min and (13.6±1.6) min. There was no significant difference between BG-PB and BG-PB-SA groups (P>0.05). (2) Compressive strength: the compressive strength of BG-PB was (16.5±1.8) MPa at 1 day and (14.1±3.7) MPa at the end of 28 days. However, the compressive strength of BG-PB-SA was (26.6±6.3) MPa on day 1 and (21.6±5.6) MPa on day 28, which was significantly higher than that of BG-PB (P<0.05). (3) pH: the pH of BG pulp capping materials' bulk immersed in simulated body fluid (SBF) went up to 8.06, and the highest pH of MTA was 8.47. Significant difference was observed between the BG pulp capping materials and MTA (P<0.05). (4) Sealing ability: the optical density (D) in positive control group was significantly higher than ln BG-PB, BG-PB-SA and MTA groups (P<0.05). And BG-PB and BG-PB-SA showed the similar favorable sealing ability with MTA, and no significant difference was observed among the three groups (P>0.05).@*CONCLUSION@#The novel BG pulp capping materials showed good physical properties, especially BG's setting time was short; BG pulp capping materials are promising.

UHPLC-LTQ-Orbitrap-based metabolomics coupled with metabolomics pathway analysis method for exploring the protection mechanism of Kudiezi injection in a rat anti-ischemic cerebral reperfusion damage model / 中国天然药物

Kudiezi injection has been used extensively in the treatment of cerebrovascular and cardiovascular diseases. However, its therapeutic effects and underlying mechanism of action are not fully understood. The aim of the present study was to clarify the protective mechanisms of Kudiezi injection on cerebral ischemic injury, using metabolomics methods. Middle cerebral artery occlusion (MCAO) was introduced in rats to build the cerebral ischemic damage. UHPLC-LTQ-Orbitrap-based analytical method was established for analysis of the metabolites. The raw mass data of all samples were normalized with Sieve 2.2 software and then introduced to orthogonal partial least squares discriminant analysis (OPLS-DA) model. Finally, 23 metabolites in plasma (15 were tentatively identified) were chosen as potential biomarkers, according to accurate mass measurements (< 5 ppm), MS/MS fragmentation patterns, and diagnostic product ions. Furthermore, on the basis of metabolic pathway analysis via metabolomics pathway analysis (MetPA), we first discovered that the protection mechanism in anti-ischemic cerebral reperfusion damage of Kudiezi injection was possibly related to the biosynthesis of phenylalanine, tyrosine, and tryptophan. The present study provided a useful approach for exploring the mechanism of ischemic stroke and evaluating the efficacy of Kudiezi injection or other traditional medicines.

HUVECs extraction and UHPLC LTQ Orbitrap analysis for screening vascular endothelium protective components in Kudiezi injection / 中国中药杂志

An effective method has been employed as a tool for screening active components in Kudiezi injection by using cell chromatography and sensitive UHPLC-HR-MSn method. The potential bioactive components in Kudiezi injection could be selectively bound to the HUVECs target cells first. After cell target desensitization and inactivation, the chemical constituents with cell target affinity were identified by LC-MS, so as to screen the possible active components in Kudiezi injection. Based on the accurate mass measurements and the retention time, in total, 9 compounds were tentatively identified and characterized, including 4 sesquiterpene lactones, 3 phenolic acids and 2 flavonoids. HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis could provide a rapid and efficient method for the identification of potential bioactive components in Kudiezi injection, and provide the reference for further research on its effective materials basis.

Rapid characterization of constituents absorbed into blood after oral administration of steroidal saponins from Radix Ophiopogonis using UHPLC-LTQ-Orbitrap MS coupled with higher energy collision induced dissociation / 药学学报

The method of UHPLC-LTQ-Orbitrap mass spectrometry coupled with higher energy collision dissociation (HCD) was established to rapidly analyze the constituents absorbed into blood after oral administration of steroidal saponins from Radix Ophiopogonis. A total of 31 constituents, including 13 furostanol steroidal saponins and 18 spirostanol steroidal saponins, were characterized based on the accurate mass measurements, fragmentation patterns, chromatographic retention times, and diagnostic product ions. Among them, 8 compounds were unambiguously identified by comparison with their corresponding standards. The results provide comprehensive insights and guidance for elucidation of material basis of Radix Ophiopogonis activity.

Influence of different drying methods on steroidal saponins and homoisoflavonoids in Ophiopogon japonicus / 中国中药杂志

To study the influence of three different drying methods (including 50 ℃-drying, 80 ℃-drying and －70 ℃-freeze-drying methods) on steroidal saponins and homoisoflavonoids in Ophiopogon japonicus,a HPLC-DAD-ELSD-MSn method was investigated to screen and identify the differential components. Through comparing the HPLC chromatograms with that of fresh O. japonicus, 50 ℃-drying medicine was similar with fresh medicine whereas the other two drying methods had great influence on the components of O. japonicus. In this study, 36 differential components were screened, among which 24 constituents(13 homoisoflavonoids and 11 steroidal saponins) were identified via HPLC-LTQ-Orbitrap MS.As a result, it was revealed that different drying methods had significant influences on the components of steroidal saponins and homoisoflavonoids. Among them, 50 ℃-drying method was the most suitable drying approach when the stability of components, cost and practicability were considered.

Rapid characterization of chemical constituents and rats metabolites of Kudiezi injection by UHPLC-LTQ-Orbitrap / 中国中药杂志

A rapid and sensitive UHPLC-HR-MSn method was used for the identification of Kudiezi injection and its main metabolites in rat plasma. After the tail intravenous injection of Kudiezi, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used, with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. Kudiezi injection and plasma were detected by ESI-LTQ-Orbitrap equipped with an ESI ion source in a negative mode. Based on the accurate mass measurements, the retention time and the mass fragmentation patterns, a total of 53 compounds were tentatively identified and characterized. Furthermore, metabolites in rat plasma after the intravenous administration of Kudiezi injection were also analyzed. A total of 38 compounds were identified, including 27 prototypes and 11 metabolites through metabolic pathways of methylation, glucuronide conjugation, sulfate conjugation and hydrolysis. As a result, UHPLC-LTQ-Orbitrap technique was applied to comprehensively expound Kudiezi injection's chemical components and constituents migrating to rat plasma, and provide scientific basis for further studies on Kudiezi injection's in vivo metabolic process and effective material base.

Effect of bioactive glasses on angiogenic growth factors of human dental pulp cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of bioactive glasses (BG) including 45S5 and nano-58S on proliferation, angiogenic markers vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion and gene expression of human dental pulp cells (HDPC).</p><p><b>METHODS</b>HDPC of 4th passage were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained 0.1 g/L 45S5 or nano-58S ionic dissolution products. Meanwhile HDPC were cultured in DMEM without BG as control group. Proliferation of the cells was evaluated with methyl thiazolyl tetrazolium (MTT) assay on day 1, 2, 3. Quantitative real-time PCR and quantitative sandwich enzyme immunoassays were used to test VEGF and bFGF gene expression and protein secretion of HDPC on day 1, 2, 3.</p><p><b>RESULTS</b>The relative growth rate (RGR) of 45S5 and nano-58S groups were (134.5 ± 5.0)% and (146.3 ± 19.8)%, which was significantly different from that of control group (P < 0.05). The quantity of VEGF secretion of two experimental groups were (189.29 ± 4.64) and (216.18 ± 14.67) ng/L, respectively, significantly higher than that of the control group [(159.03 ± 11.69) ng/L] (P < 0.05). Furthermore, the nano-58S group secreted much more VEGF than 45S5 group (P < 0.05).bFGF secretion of HDPC was also enhanced by both 45S5 and nano-58S bioactive glasses. The VEGF gene expression of 45S5 and nano-58S on day 1 were (1.70 ± 0.19) and (1.63 ± 0.42), while the bFGF gene expressin on day 3 were (1.49 ± 0.02) and (2.30 ± 0.04), all significantly higher than that of control group (P < 0.05).</p><p><b>CONCLUSIONS</b>Bioactive glasses can enhance the proliferation, VEGF and bFGF secretion and gene expression of human dental pulp cells. Compared with 45S5, nano-58S showed a higher activation.</p>