Punicalagin Ameliorates Diabetic Liver Injury by Inhibiting Pyroptosis and Promoting Autophagy via Modulation of the FoxO1/TXNIP Signaling Pathway.

Diabetic liver injury (DLI) is one of the complications of diabetes mellitus, which seriously jeopardizes human health. Punicalagin (PU), a polyphenolic compound mainly found in pomegranate peel, has been shown to ameliorate metabolic diseases such as DLI, and the mechanism needs to be further explored. In this study, a HFD/STZ-induced diabetic mouse model is established to investigate the effect and mechanism of PU on DLI. The results show that PU intervention significantly improves liver histology and serum biochemical abnormalities in diabetic mice, significantly inhibits the expression of pyroptosis-related proteins such as NLRP3, Caspase1, IL-1ß, and GSDMD in the liver of diabetic mice, and up-regulated the expression of autophagy-related proteins. Meanwhile, PU treatment significantly increases FoxO1 protein expression and inhibits TXNIP protein expression in the liver of diabetic mice. The above results are further verified in the HepG2 cell injury model induced by high glucose. AS1842856 is a FoxO1 specific inhibitor. The intervention of AS1842856 combined with PU reverses the regulatory effects of PU on pyroptosis and autophagy in HepG2 cells. In conclusion, this study demonstrates that PU may inhibit pyroptosis and upregulate autophagy by regulating FoxO1/TXNIP signaling, thereby alleviating DLI.

Factors Associated with Functional Constipation among Students of a Chinese University: A Cross-Sectional Study.

Functional constipation (FC) is prevalent worldwide and is an increasingly prominent problem among university students. However, there is a paucity of research on FC in university students. This study aimed to assess the prevalence of FC among Chinese university students by the Rome III criteria and investigate its associated factors. This cross-sectional study was conducted by online questionnaires among 929 university students at a Chinese university. Food consumption was assessed with the Semi-Quantitative Food Frequency Questionnaire (SQFFQ) and dietary patterns were analyzed using factor analysis. A binary logistic regression model was applied to clarify FC-associated factors. The prevalence of FC among university students was 5.1%. Interestingly, among university students, the prevalence of FC with "complex" dietary pattern was significantly higher than those with "vegetable, fruit, egg and milk-based" and "livestock and aquatic product-based" dietary pattern (9.9% vs. 3.1% vs. 2.8%, p < 0.001). The prevalence of FC was significantly higher among university students with moderate to severe sleep disorders than those with the other sleep status (χ2 = 18.100, p < 0.001). Furthermore, after adjusting the covariates, "complex" dietary pattern (OR = 4.023, p < 0.001), moderate to severe sleep disorders (OR = 3.003, p = 0.006), overeating (OR = 2.502, p = 0.032), long mealtime (>30 min) (OR = 6.001, p = 0.007), and poor defecation habits (OR = 3.069, p = 0.042) were positively associated with FC among university students. Based on the above-associated factors for FC, improving dietary patterns and sleep status and developing good bowel and dietary habits are essential to prevent and alleviate university students' FC.

Punicalagin Protects against Diabetic Liver Injury by Upregulating Mitophagy and Antioxidant Enzyme Activities.

Diabetic liver injury has received increasing attention as a serious complication of type 2 diabetes. Punicalagin (PU), a major component of pomegranate polyphenols, has various biological activities such as antioxidant, anti-inflammatory, and lipid metabolism regulation. In this study, we observed the protective effect of punicalagin on a high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic liver injury in mice and revealed the underlying mechanism. The results showed that fasting blood glucose (FBG), fasting serum insulin (FINS), and homeostasis model assessment for insulin resistance (HOMA-IR) in diabetic liver injury mice were significantly decreased after punicalagin intervention. Simultaneously, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), free fatty acids (FFA), malondialdehyde (MDA), and total superoxide dismutase (T-SOD) in the serum and liver were significantly decreased, with reductions in fat lesions and inflammatory cells. Mitophagy is a selective autophagy that maintains a balance between the quality and quantity of intracellular mitochondria. Studies have shown that mitophagy is closely related to the occurrence and development of diabetic liver injury. In our study, the mitochondrial membrane potential (MMP) was significantly increased in mice with diabetic liver injury after punicalagin intervention; the protein expression of Pink1, Parkin, Bnip3, LC3b, P62, manganese superoxide dismutase (MnSOD), and catalase (CAT) was significantly increased in the liver; and the activities of MnSOD and CAT in the serum and liver were significantly increased, which is consistent with the results of in vitro experiments. In summary, our study provided evidence that punicalagin could reduce the level of oxidative stress in the liver by upregulating mitophagy and the activities of antioxidant enzymes, thus having a certain protective effect against diabetic liver injury.

Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway.

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice.

We report here a modified auxotrophic strain of Salmonella typhimurium that can target and cure breast tumors in orthotopic mouse models. We have previously reported development of a genetically modified strain of S. typhimurium, selected for prostate tumor targeting and therapy in vivo. The strain, termed S. typhimurium A1, selectively grew in prostate tumors in xenograft models causing tumor regression. In contrast, normal tissue was cleared of these bacteria even in immunodeficient athymic mice with no apparent side effects. A1 is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support only from tumor tissue. The ability to grow in viable tumor tissue may account, in part, for the unique antitumor efficacy of the strain. In the present report, to increase tumor-targeting capability of A1, the strain was reisolated after infection of a human colon tumor growing in nude mice. The tumor-isolated strain, termed A1-R, had increased targeting for tumor cells in vivo as well as in vitro compared with A1. Treatment with A1-R resulted in highly effective tumor targeting, including viable tumor tissue and significant tumor shrinkage in mice with s.c. or orthotopic human breast cancer xerographs. Survival of the treated animals was significantly prolonged. Forty percent of treated mice were cured completely and survived as long as non-tumor-bearing mice. These results suggest that amino acid auxotrophic virulent bacteria, which selectively infect and attack viable tumor tissue, are a promising approach to cancer therapy.

Homogeneous, nonradioactive, enzymatic assay for plasma pyridoxal 5-phosphate.

BACKGROUND: Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B(6). Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely diagnosed because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the PLP-dependent recombinant enzyme, homocysteine-alpha,gamma-lyase (rHCYase). METHODS: PLP was removed from holoenzyme rHCYase by incubation with hydroxylamine to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme was linearly related to the amount of PLP bound to the enzyme. The amplification principle of the assay allowed nanomolar concentrations of PLP to be measured by the conversion (by reconstituted holo-rHCYase) of millimolar concentrations of homocysteine to H(2)S. N,N-Dibutylphenylenediamine (DBPDA) was used for determination of H(2)S, the combination of which forms a chromophore with high absorbance. The assay was initiated by incubation of 5 microL of plasma with apo-rHCYase in a binding buffer for 60 min at 37 degrees C. Homocysteine (2.5 mmol/L) was added to the assay buffer and incubated at 37 degrees C for 20 min. The DBPDA reaction was allowed to progress for 10 min and then read at 675 nm. RESULTS: The PLP enzymatic assay has a lower limit of detection of 5 nmol/L and is linear to 200 nmol/L. The recovery of PLP was 98%. The mean within- and between-run CVs were 9.6% and 12%, respectively. Correlation of 45 samples in the PLP enzymatic assay and the B(6) (3)H radioenzymatic assay (American Laboratory Products Co., Ltd.) yielded: y = 0.9367x + 10.569 (R(2) = 0.9201). CONCLUSIONS: This new PLP assay is the first homogeneous, nonradioactive, vitamin B(6) diagnostic method. The assay is applicable to chemistry automated analyzers and may have wide clinical use.