Quercetin-Rich Guava (Psidium guajava) Juice in Combination with Trehalose Reduces Autophagy, Apoptosis and Pyroptosis Formation in the Kidney and Pancreas of Type II Diabetic Rats

Abstract

We explored whether the combination of anti-oxidant and anti-inflammatory guava (Psidium guajava) and trehalose treatment protects the kidney and pancreas against Type II diabetes (T2DM)-induced injury in rats. We measured the active component of guava juice by HPLC analysis. T2DM was induced in Wistar rats by intraperitoneal administration of nicotinamide and streptozotocin and combination with high fructose diets for 8 weeks. The rats fed with different dosages of guava juice in combination with or without trehalose for 4 weeks were evaluated the parameters including OGTT, plasma insulin, HbA1c, HOMA-IR (insulin resistance) and HOMA-β (β cell function and insulin secretion). We measured oxidative and inflammatory degrees by immunohistochemistry stain, fluorescent stain, and western blot and serum and kidney reactive oxygen species (ROS) by a chemiluminescence analyzer. High content of quercetin in the guava juice scavenged H2O2 and HOCl, whereas trehalose selectively reduced H2O2, not HOCl. T2DM affected the levels in OGTT, plasma insulin, HbA1c, HOMA-IR and HOMA-β, whereas these T2DM-altered parameters, except HbA1c, were significantly improved by guava and trehalose treatment. The levels of T2DM-enhanced renal ROS, 4-hydroxynonenal, caspase-3/apoptosis, LC3-B/autophagy and IL-1β/pyroptosis were significantly decreased by guava juice and trehalose. The combination with trehalose and guava juice protects the pancreas and kidney against T2DM-induced injury.

Keywords: Type II diabetes; apoptosis; autophagy; oxidative stress; pyroptosis; quercetin; trehalose.

Figure 1

Typical chromatogram of quercetin content in the extract of guava by HPLC analysis. (A) The chromatogram of the standard mixture is shown in the left panel (A), whereas the chromatogram of the water guava extract is indicated in the right panel (B); Five chemicals including ellagic acid, ferulic acid, rosmarinic acid, quercetin and narinegenin are identified in the standard mixture. Only quercetin is found in the guava juice; (C) Quercetin content in the extract of guava juice by HPLC analysis.

Figure 2

The effect of different concentration of guava juice (A,B), trehalose (C,D) and different kinds of guava extraction (E,F) on scavenging ROS levels of H₂O₂ and HOCl in vitro. n = 3–5 in each group. Water extract of Thailand guava, GWE1; Water extract of pearl guava, GWE2; ethanol extract of Thailand guava, GEE1; ethanol extract of pearl guava, GEE2. * p < 0.05 vs. H₂O. # p < 0.05 GEE1 vs. GWE1.

Figure 3

Effect of guava juice and trehalose on glycemic parameters. (A) Effect of guava juice and trehalose on oral glucose tolerance test. Normal rats were orally intake 40% guava juice containing 12% trehalose (red), 8% sucrose (black) or ddH₂O (Blank, white); (B) Intravenous glucose and trehalose tolerance test. Normal animals were given 0.5 g/kg glucose (black) or trehalose (white) via an intravenous route. n = 8 in each group; (C) HbA1c Level. n = 3–5 in each group; (D–I) Oral glucose tolerance test. n = 3–5 in each group. Data are expressed as mean ± SEM. * p < 0.05 vs. CON (control group).

Figure 4

Effect of guava juice and trehalose on OGTT (A); insulin levels (B); HOMA IR (C) and HOMA-β (D) in six groups of rats at week 0 and week 4. Data are expressed as mean ± SEM and analyzed by two-way ANOVA. n = 3–5 in each group. * p < 0.05 vs. the value at week 0 of respective group. # p < 0.05 vs. DM.

Figure 5

The typical graphs of H & E (left two panels) and Masson’s stains (right two panels) in the pancreas and kidney from six groups of rats.

Figure 6

The typical graph of expression (brown area indicated by arrows) of 4-HNE, IL-1β, caspase 3 in renal glomeruli and tubules, and fluorescent LC3-B (red color) and caspase 3 (green color) in renal tubules of six groups (A). The statistical data of 4-HNE (B); IL-1β (C); caspase 3 (D) and LC3-B (E) are demonstrated in these six groups; (F) The original curves show the real-time data of renal ROS during 7200 s of recording in 6 groups of rat kidneys. Statistic data of renal ROS in vivo (G) and serum ROS levels in vitro (H) are indicated. Data are expressed as mean ± SEM. n = 3–5 each group. * p < 0.05 vs. CON, # p < 0.05 vs. DM, ^a p < 0.05 vs. B1.* p < 0.05 vs. CON. # p < 0.05 vs. DM. ^a p < 0.05 vs. B1.

Figure 7

The typical graph of pancreatic expression (brown area indicated by arrows) of 4-HNE (lipid peroxidation and oxidative stress marker), IL-1β (pyroptosis marker), caspase 3 (apoptosis marker) and fluorescent LC3-B (red color, autophagy marker) and caspase 3 (green color) in pancreas of six groups. Data are expressed as mean ± SEM. n = 3–5 each group. * p < 0.05 vs. CON. # p < 0.05 vs. DM. ^a p < 0.05 vs. B1.

Figure 8

Effect of guava juice and trehalose on apoptosis-, autophagy- and pyroptosis-related proteins expression in the T2DM kidney and pancreas by western blot. Typical western blotting graphs of kidney (A) and pancreas (I) are shown; The statistical data of each protein expression and ratio are shown in the kdieny (B–H) and pancreas (J–P). Data are expressed as mean ± SEM. n = 3–5 each group.* p < 0.05 vs. CON. # p < 0.05 vs. DM. ^a p < 0.05 vs. B1.