| Summary: | 碩士 === 弘光科技大學 === 生物科技研究所 === 94 === In this study, we used glucose (G), and dicarbonyl compounds﹝glyoxal (GO), and methylglyoxal (MGO)﹞to perform the in vitro studies on the glycative reactions between the inducers (G, GO, and MGO) and bovine serum albumin (BSA), plasma and/or low-density lipoprotein (LDL), taking the advantage of the fluorescence emission to estimate the extent of advanced glycation end products (AGEs) production. In the meanwhile, in the preventive strategy for AGEs formation, we tried several herbal aqueous extracts including those from Momordica charantia L. (ME), Graptopetalum paragugayene E. Walther (GE), Psidium guajava L. (PE), and Toona sinensis Roem (TE). In addition, adopting the DPPH radical scavenging capability (DRSC) of the polyphenolics and the total flavonoids, we tried to understand the correlation between such DRSC of each individual extract, or the active constituent if any, with the antiglycative potentiality. As a final result, PE was selected as the most potent among all. By which we studied the protective effect of PE on the damages caused by G, GO, and MGO on the cell line human umbilical vein endothelial cells (HUVEC), such as apoptotic effects and its related molecular mechanism. We also examined the active constituents in the PE, as a combination to interpret the molecular action mechanism of PE.
Results indicated that for the inducer G, the AGEs in BSA and plasma were reduced to 40 % by PE at 0.5 mg/mL, while revealing 40~60 % of antiglycative effect on those induced by GO and MGO. On the other hand, TE, ME, and GE at 0.5 mg/mL showed 59 %, 24 %, and 31 % of inhibitory effect on glycation induced by glucose in plasma, respectively. PE had very prominent anti-LDL glycative activity as well as for protein glycations. PE effectively exhibited 63.45±5.80 %, and 36.58±4.55 % efficiency on AGEs formation induced by G, and GO, respectively. The lag times for conjugated diene (CD) formation (in min) were increased 6.4 and 5.2-folds in the presence of inducers G and GO, respectively. As for the anti-thiobarbituric acid reactive substances (TBARS) efficiency (in %), PE showed 75.77±0.19 % at 0.5 mg/mL, when induced by G; and 36.68 ±1.01 % induced by GO. The REM, an indicator of LDL oxidative extent, was 1.6, and 2.0 for G and GO with respect to PE (0.1 mg/mL) compared to the control (4.2, and 4.0), respectively. PE was the best free radical scavenging species for the DPPH free radicals, the order of superiority was PE> TE > ME > GE. PE was also prominent for its rich total polyphenolic content (156.61±10.39 mg/g), with the order in decreasing level: PE (156.61±10.39 mg/g)>TE (79.90±2.05 mg/g)>ME (978.30±5.43 mg/g)>GE (20.81±6.24 mg/g). Similarly, PE was also enriched with the highest flavonoid content (82.85±0.22 mg/g) among these four medicinal herbs. Hence PE was speculated to be potentially effective also for pro-diabetic complications, especially the cardiovascular damages induced by ROS.
In order to understand the molecular action mechanism for PE acting as an effective antiglycative agent, we further investigated the effects of G, GO, and MGO on the antiproliferation, apoptosis, signal transduction pathway, the generation of reactive oxygen species (ROS), and released nitric oxide (NO) in HUVEC as well. The methodologies used involved the cell viability assay (MTT assay) and the LDH releasing capability. In this respect, we used G (30 mM), GO (0.5 mM) and MGO (0.5 mM) to induce such damages as mentioned on the cell line HUVEC. G, GO, and MGO decreased cell viability of HUVEC to 1.38, 1.83, and 1.29 %, respectively. In contrast, the LDH release caused by G, GO, and MGO was significantly different as compared with the control in HUVEC, changing from 22.22 to 82.22 %, 22.22 to 85.32 % and 22.22 to 76.23 %, respectively in the presence of G, GO and MGO, respectively. On the other hand, the speculation that the cells might have been damaged in the high osmotic pressure environment as in G (30 mM), the cells were incubated under controlled conditions with high concentration of mannitol (an osmotic control), results did not show significant damaging effect. In cell viability assay, PE at 5 μg/mL, 100 μg/mL, and 100 μg/mL revealed the inhibition capability, respectively against the inducers G, GO, and MGO. The inhibitory effect of PE on the cell mortality induced by G at a concentration of 5 μg/mL was shown to change from 62.46 % to 75.71 %. And that induced by G, and MGO with a concentration of PE 100 μg/mL was shown to change from 73.68 % to 99.60 %, and 69.82 % to 93.92 %, respectively. As for LDH release test, PE at 50 μg/mL, 2 μg/mL, and 100 μg/mL revealed the inhibition capability respectively against the inducers G, GO, and MGO. The inhibitory effect of PE on the LDH release was shown to have reduced from 82.22 % to 76.67 %, 85.32 % to 78.32 %, and 76.23 % to 66.64 %, respectively in case of G, GO and MGO. Thus LDH release and cell mortality of HUVEC caused by the glycative inducers G, GO and MGO was shown to have been suppressed by PE. To seek the cause of reduction in viability as affected by the apoptotic effect. We quantified the degree of apoptosis: the amount of sub-G1 DNA was analyzed by flow cytometry. Flow cytometric analysis indicated that HUVEC when exposed to G, GO, and MGO had significantly elevated apoptotic effect. The apoptosis cell was 1.00 %, 7.18 %, and 21.16 % with respect to inducers compared to the blank (0.20 %). The inhibitory effect of PE on the apoptotic effect induced by G, GO, and MGO was shown to reach 1.00 % to 0.57 %, 7.18 % to 1.26 %, and 21.16 % to 6.49 %. In HUVEC, the expression of matrix metalloproteinase-2 (MMP-2) was elevated to 5.89, 3.36, and 2.15-folds in the presence of the inducer G, GO, and MGO, respectively. Expressions of MMP-2 induced by G, GO, and MGO were significantly inhibited with efficiency of 62.49 %, 61.64 % and 82.86 %.
The activation of signal transduction pathway was also studied to access the inhibitory effect of PE at 100μg/mL. We determined the mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and the nuclear factor κB (NFκB) in cell line HUVEC when incubated with G, GO and MGO. PE has shown to have inhibited the phospho-p38-MAPK activity induced by G. The phospho-JNK-MAPK, and phospho-ERK-MAPK activity were also significantly inactivated by PE comparing with the control. The examination of phospho-p38-MAPK, and phospho-ERK-MAPK revealed that GO significantly elevated both the activities of phospho-p38-MAPK and phospho-ERK-MAPK. PE was able to down regulate the expression of phospho-p38-MAPK and phospho-ERK-MAPK induced by GO, and MGO, respectively. Alternatively, PKC was significantly activated in G, GO, and MGO-treated HUVEC cells, and PE has shown significant inhibitory effect on PKC activity induced by G. Moreover, PE also effectively inhibited with 5.76 %, 14.34 %, and 16.27 % efficiency on NFκB activity induced by G, GO, and MGO, respectively.
Yet, it is worth to understand whether the generation of oxidative stress could affect apoptosis and the signal transduction pathway in diabetic vessels and endothelial cells when cells were cultivated with G, GO, and MGO. The detection of ROS generation and LDH release were thus further performed. The ROS generation was significantly increased in G, GO, and MGO-treated HUVEC. PE effectively inhibited with 57.83 %, 55.76 % and 44.44 % efficiency on the ROS generation induced by G, GO, and MGO, respectively. The NO release was significantly increased in G, GO, and MGO-treated HUVEC. PE effectively inhibited the NO release induced respectively by G, GO, and MGO, reaching an efficiency of 90.97, 88.76 and 84.17 %, respectively. Our data has indicated that the peroxynitrite derived from the ROS and superoxide anion reacting with NO is the main apoptotic factors that can be induced in the presence of high concentrations of G, GO, and MGO, implicating the fact that PE is effective in suppressing the oxidative stress derived from the glycation.
The main phenolic compounds found in guava leaf extract were quercetin (12.26 mg/g), gallic acid (12.18 mg/g) and ferulic acid (9.42 mg/g). MGO inhibited significantly antithrombinⅢ (ATⅢ) activity, while PE displayed as an effective protective agent against MGO-induced inactivation of ATⅢ. An additive effect was seen when these three phenolics were used simultaneously, a 85 % recovery was seen for the antithrombin Ⅲ activity caused by MGO. Thus the preliminary results have revealed the antiglycative activity and the free radical scavenging capability can be ascribed to the enriched contents of polyphenolics and flavonoids in PE.
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