Mechanism of Gegen Qinlian Decoction in improving glucose metabolism in vitro and in vivo by alleviating hepatic endoplasmic reticulum stress / 中国中药杂志

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.

Phagocytic responses of peritoneal macrophages and neutrophils are different in rats following prolonged exercise

OBJECTIVE: To analyze the effects of exhausting long-duration physical exercise (swimming) sessions of different durations and intensities on the number and phagocytic capacity of macrophages and neutrophils in sedentary rats. INTRODUCTION: Exercise intensity, duration and frequency are important factors in determining immune response to physical effort. Thus, the effects of exhausting long-duration exercise are unclear. METHODS: Wistar rats were divided into two groups: an untreated group (macrophage study) and oyster glycogen-treated rats (neutrophil study). In each group, the animals were subdivided into five groups (10 rats per group): unexercised controls, an unadapted low-intensity exercise group, an unadapted moderate-intensity exercise group, a preadapted low-intensity exercise group and a preadapted moderate-intensity exercise group. All exercises were performed to exhaustion, and preadaptation consisted of 5, 15, 30 and 45 min sessions. RESULTS: Macrophage study: the number of peritoneal macrophages significantly decreased (9.22 ± 1.78 x 10(6)) after unadapted exercise but increased (21.50 ± 0.63 x 10(6)) after preadapted low-intensity exercise, with no changes in the moderate-intensity exercise group. Phagocytic capacity, however, increased by more than 80 percent in all exercise groups (low/moderate, unadapted/preadapted). Neutrophil study: the number of peritoneal neutrophils significantly decreased after unadapted (29.20 ± 3.34 x 10(6)) and preadapted (50.00 ± 3.53 x 10(6)) low-intensity exercise but increased after unadapted (127.60 ± 5.14 x 10(6)) and preadapted (221.80 ± 14.85 x 10(6)) moderate exercise. Neutrophil phagocytic capacity decreased by 63 percent after unadapted moderate exercise but increased by 90 percent after corresponding preadapted sessions, with no changes in the low-intensity exercise groups. CONCLUSION: Neutrophils and macrophages of sedentary rats respond differently to exercise-induced stress. Adaptation sessions reduce exercise-induced stress on the immune system.

A simple quantitative in vitro macrophage migration assay.

An in vitro macrophage chemotaxis model using mouse peritoneal non-elicited resident macrophage cells and chemotaxins containing mediators of non-specific elicitors such as oyster glycogen or sodium caseinate has been described. Macrophage cells accumulation in mouse peritoneal cavity was maximum at 48 hr after injecting (i.p.) oyster glycogen (2.5%) or sodium caseinate (12%), 0.5 ml/mouse. Chemotaxins containing mediators were prepared from these mice by peritoneal lavage and termed as routine 'diluted' cocktail and 'concentrated (3 times)' cocktail. Chemotaxis assays were carried out in a modified Boyden chamber using a 48-well microchemotaxis assembly. In vitro results showed higher macrophage chemotaxis response against the 'concentrated' cocktails as compared to routine 'diluted' cocktail. Macrophages exhibited cell density dependent increase in the responsiveness to chemoattractant and macrophage cell density of 4 x 10(6) per ml concentration in the upperwell was found to be optimum. Macrophage responsiveness was seen better with sodium caseinate cocktail as compared to oyster glycogen in vitro as well as in vivo. DMSO (Dimethyl Sulphoxide) solvent (0.25% conc.) did not interfere with normal macrophage chemotaxis. Both CO2 incubator (5% CO2 in air) and BOD incubator with humidified chamber favoured chemotaxis. In vitro test system described can be used as a model to study the effect of anti-inflammatory compounds directly on the macrophage chemotaxis.