The Classification, Molecular Structure and Biological Biosynthesis of Flavonoids, and Their Roles in Biotic and Abiotic Stresses.

With the climate constantly changing, plants suffer more frequently from various abiotic and biotic stresses. However, they have evolved biosynthetic machinery to survive in stressful environmental conditions. Flavonoids are involved in a variety of biological activities in plants, which can protect plants from different biotic (plant-parasitic nematodes, fungi and bacteria) and abiotic stresses (salt stress, drought stress, UV, higher and lower temperatures). Flavonoids contain several subgroups, including anthocyanidins, flavonols, flavones, flavanols, flavanones, chalcones, dihydrochalcones and dihydroflavonols, which are widely distributed in various plants. As the pathway of flavonoid biosynthesis has been well studied, many researchers have applied transgenic technologies in order to explore the molecular mechanism of genes associated with flavonoid biosynthesis; as such, many transgenic plants have shown a higher stress tolerance through the regulation of flavonoid content. In the present review, the classification, molecular structure and biological biosynthesis of flavonoids were summarized, and the roles of flavonoids under various forms of biotic and abiotic stress in plants were also included. In addition, the effect of applying genes associated with flavonoid biosynthesis on the enhancement of plant tolerance under various biotic and abiotic stresses was also discussed.

Genome-wide identification and characterization of PdbHLH transcription factors related to anthocyanin biosynthesis in colored-leaf poplar (Populus deltoids).

Basic helix-loop-helix (bHLH) proteins are transcription factors (TFs) that have been shown to regulate anthocyanin biosynthesis in many plant species. However, the bHLH gene family in Populus deltoids has not yet been reported. In this study, 185 PdbHLH genes were identified in the Populus deltoids genome and were classified into 15 groups based on their sequence similarity and phylogenetic relationships. Analysis of the gene structure, chromosome location and conserved motif of the PdbHLH genes were performed by bioinformatic methods. Gene duplication analyses revealed that 114 PdbHLH were expanded and retained after WGD/segmental and proximal duplication. Investigation of cis-regulatory elements of PdbHLH genes indicated that many PdbHLH genes are involved in the regulation of anthocyanin biosynthesis. The expression patterns of PdbHLHs were obtained from previous data in two colored-leaf poplar (QHP and JHP) and green leaf poplar (L2025). Further analysis revealed that 12 candidate genes, including 3 genes (PdbHLH57, PdbHLH143, and PdbHLH173) from the subgroup III(f) and 9 gene from other groups, were positively associated with anthocyanin biosynthesis. In addition, 4 genes (PdbHLH4, PdbHLH1, PdbHLH18, and PdbHLH164) may be involved in negatively regulating the anthocyanin biosynthesis. These results provide a basis for the functional characterization of bHLH genes and investigations on the molecular mechanisms of anthocyanin biosynthesis in colored-leaf poplar.

[Involvement of Wnt/ß-catenin signaling in the osteogenesis of bone marrow mesenchymal stem cells induced by drynaria total flavonoids].

OBJECTIVE: To explore the effects of the expression of Wnt/ß-catenin signaling factor mRNA during drynaria total flavonoids on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: The BMSCs were isolated from SD rats by whole bone marrow culture method and purified by passage. And the P3 BMSCs were intervened with 100 µg/ml drynaria total flavonoids. At Day 21, mineralized staining was performed. At Days 7, 14, 21 and 28 post-intervention, the activity of alkaline phosphatase (ALP) was detected and polymerase chain reaction (PCR) used to detect the expressions of Wnt/ß-catenin signaling pathway related factors ß-catenin, LEF-1 and cycline D mRNA. RESULTS: At each time point post-intervention, comparing the ALP activity in cell supernatant between two group, the drynaria total flavonoids group was higher than the blank control group (7 d: 11.10 ± 0.08 vs 1.61 ± 0.14; 14 d: 24.62 ± 0.34 vs 1.64 ± 0.04; 21 d: 18.41 ± 0.06 vs 1.53 ± 0.04; 28 d: 14.9 ± 0.14 vs 1.52 ± 0.04; all P < 0.01). At Day 21, upon staining with alizarin red, the drynaria total flavonoids group was positive while the blank control group negative. At Day 14, the expression of ß-catenin mRNA was higher in the drynaria total flavonoids group higher than that in the blank control group (0.357 ± 0.063 vs 0.174 ± 0.013, P < 0.05). At Day 7, the expressions of LEF-1 and cycline D mRNA were higher in the drynaria total flavonoids group than those in the blank control group (LEF-1 0.0611 ± 0.0002 vs 0.0345 ± 0.0131; cycline D 0.1510 ± 0.0255 vs 0.0718 ± 0.0294, all P < 0.05). CONCLUSION: Drynaria total flavonoids induce BMSCs to differentiate into osteoblasts. And it is accompanied with the altered expression of Wnt/ß-catenin signaling pathway related factor mRNA.

Effect of an Ilex asprella root decoction on the related genes of lipid metabolism from chronic stress and hyperlipidemic fatty liver in rats.

BACKGROUND: The gradually increasing changes in a human hyperlipidemic diet along with chronic stress might play an important role in the increased numbers of fatty liver. This study investigated the effects of Ilex asprella root decoction on related genes of lipid metabolism in chronic stress in hyperlipidemic fatty liver in rats. METHODS: Forty-eight male Wistar rats were randomly divided into four groups: normal control group, model control group, simvastatin group, and Ilex asprella root group. To establish chronic stress and hyperlipidemic fatty liver models in rats, the levels of serum lipids, glucose, liver index, insulin (INS), insulin resistant (IR) index, adiponectin, superoxide dismutase (SOD), glutathione peroxidase (GSH-pX), glutathione (GSH), liver X receptor (LXR), and sterol responsive element binding protein (SREBP)-1c in rats were measured. RESULTS: When compared to the normal control group, the levels of serum lipids, glucose, liver index, INS, IR index, and GSH in the model control group significantly increased (P < 0.01). The protein levels of LXRα and SREBP-1c increased (P < 0.05), and the serum adiponectin and the SOD and GSH-pX decreased significantly (P < 0.01). When compared to the model control group, the levels of serum lipids, glucose, liver index, INS, IR index, SOD, and GSH-pX in the simvastatin group and Ilex asprella root group increased in varying degrees (P < 0.01 or 0.05); the serum adiponectin and GSH decreased (P < 0.05), while the protein levels of LXRα and SREBP-1c decreased in varying degrees (P < 0.01 or 0.05). When compared to the simvastatin group, the IR index and protein levels of LXRα in the Ilex asprella root group decreased (P < 0.05), and the serum adiponectin and SOD increased (P < 0.05). CONCLUSION: The Ilex asprella root decoction has some protective effects on regulating the related genes of lipid metabolism caused by chronic stress and hyperlipidemic fatty liver in rats.

[Relationship of glucose metabolic rate and plasma levels of adiponectin and leptin in patients with metabolic syndrome].

OBJECTIVE: To investigate the relationship of glucose metabolic rate (GMR) and plasma levels of adiponectin and leptin in patients with metabolic syndrome (MS). METHODS: A total of 30 MS subjects aged 36-60 years old were selected as MS group. And 20 normal adults were selected as control group. The GMR was evaluated by the technique of hyperinsulinemic euglycemia clamp. The plasma concentrations of adiponectin and leptin were detected by enzyme-linked immunosorbent assay (ELISA). Blood pressure, waist circumference (WC), body weight and body height were measured. RESULTS: (1) During the steady state (last 30 min), the GMR was significantly lower in MS group than that in control Group [(4.13 ± 1.34) mg×kg(-1)×min(-1) vs (8.33 ± 1.59) mg·kg(-1)×min(-1), P < 0.01]. (2) The plasma level of adiponectin was significantly lower in MS group than that in control group [(5.15 ± 2.54) µg/ml vs (10.28 ± 5.50) µg/ml, P < 0.01]. The plasma level of leptin were significantly higher in MS group than that in control group [(189.37 ± 90.48) ng/ml vs (126.55 ± 72.70) ng/ml, P < 0.01]. (3) In MS group, glucose metabolic rate was associated with WC, BMI, TG, HDL-C FINS, leptin, and adiponectin, (all P < 0.05). CONCLUSION: The technique of hyperinsulinemic euglycemic clamp shows that the BMR of MS patients significantly decreases. It may be associated with their lowered plasma levels of adiponectin and leptin.

[In vitro studies on the growth and proliferation characteristics of rat bone mesenchymal stem cells].

OBJECTIVE: This study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease. METHODS: Bone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates. RESULTS: Flow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day. CONCLUSION: The whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.

[Effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells at different glucose concentrations: experiment with rats].

OBJECTIVE: To study the effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at different glucose concentrations. METHODS: BMSCs of SD rats were isolated, cultivated in vitro, and divided into 6 groups to be induced to differentiate into osteoblasts under different conditions: (1) low glucose control group, (2) high glucose control group, (3) low glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (4) high glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (5) low glucose+drynaria total flavonoid group, and (6) high glucose with drynaria total flavonoid group. Alkaline phosphate (ALP) test kit was used to examine the level of ALP. The ALP staining positive rate was examined with modified calcium cobalt method. Alizarin red staining was adopted to observe the number of calcium nodes. Immunohistochemistry was used to detect type I collagen level. Advanced glycosylation end products (AGEs) were tested by ELISA. RESULTS: The A value indicating the ALP activity, ALP staining positive rate, calcium node number, and type I collagen expression score of the low glucose+drynaria total flavonoid group were (0.439±0.024), 48.7%, (9.75±1.71) nodes/HP, and (2.21±0.07) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.385±0.029), 35.0%, (6.25±0.96) nodes/HP, and (1.93±0.13) respectively, all P<0.05]. The A value, ALP staining positive rate, calcium node number, and type I collagen expression score of the high glucose with drynaria total flavonoid group were (0.352±0.022), 25.3%, (4.50±1.29)/HP, and (1.70±0.03) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.139±0.013), 22.7%, (3.25±1.50)/HP, and (1.28±0.27) respectively, all P<0.05]. The AGE expression levels of the high glucose classical induction group and high glucose+drynaria total flavonoid group were both significantly higher than those of the low glucose classical induction group and low glucose+drynaria total flavonoid group (both P<0.05). There were no significant differences in the AGE level among the low glucose control, low glucose classical induction, and low glucose+drynaria total flavonoid groups (all P<0.05); and among the high glucose control, high glucose classical induction, and high glucose+drynaria total flavonoid groups (all P<0.05). However, the AGE levels of the high glucose groups were all significantly higher than those of the corresponding low glucose groups (all P<0.05). Glucose increased the AGE levels dose- and time-dependently. The concentrations of AGEs were significantly negatively correlated with the expression of type I collagen (r=-0.410, P<0.05). CONCLUSIONS: Drynaria total flavonoid promotes the osteogenic differentiation of BMSCs and relieves the inhibitory effect of osteogenic differentiation by glucose at high concentration. Thus drynaria total flavonoid may provide a potential therapy for diabetic osteoporosis.

[Effect of TIEG-siRNA on Smad2, p-smad2 and collagen IV in diabetic nephropathy].

OBJECTIVE: To investigate the effect of TIEG-siRNA on Smad2, p-smad2 and collagen IV in diabetic nephropathy rats induced by streptozotocin (STZ). METHODS: Ten Sprague-Dawley rats injected with STZ were randomly divided into TIEG-siRNA and Control groups. Other five normal rats were used as control. Each of the TIEG-siRNA and Control groups were injected with TIEG-siRNA and Control 0.5 ml via tail vein at 0 and 72 hours respectively. All rats were sacrificed at week 4 after a successful modeling. To confirmed the efficacy of TIEG-siRNA in rat kidney, the TIEG levels were determined by fluorescence quantitative PCR. The expressions of Smad2, p-smad2 and collagen IV protein were detected by immunohistochemical method while Smad2 and p-smad2 examined by Western blot. RESULTS: The TIEG levels were greatly down-regulated in the TIEG-siRNA treated group (0.0636 ± 0.0066) versus the empty vector treated group (0.1054 ± 0.0111) (P < 0.05). The immunohistochemical semi-quantitative method indicated that there was a decrease in the TIEG-siRNA treated group versus the empty vector treated group: (2.13 ± 0.19)% vs (2.53 ± 0.34)% in Smad2, (21.77 ± 2.00)% vs (27.03 ± 2.51)% in p-smad2 and (3.67 ± 0.42)% vs (4.85 ± 0.43)% in collagen IV. Western blot also showed that smad2 in the TIEG-siRNA treated group (0.32 ± 0.09) was much lower than that in the empty vector treated group (0.50 ± 0.04). And p-smad2 in the TIEG-siRNA treated group (0.16 ± 0.01) was much lower than that in the empty vector treated group (0.32 ± 0.02) (P < 0.05). CONCLUSION: TIEG-siRNA may be useful in preventing the progression of diabetic nephropathy through influencing the expression of Smad2 and its activation and down-regulating collagen IV.