Effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate the systemic inflammatory response in acute pancreatitis

This research was carried out to investigate the effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate systemic inflammatory response in acute pancreatitis. In this study, the rat pancreatic acini AR42J cells were chosen as the research object, the application of hyla induced pancreatic acinar cells made model for acute pancreatitis, application of different concentrations of angelica peony spread effect on building cells, thus divided into control group, built in the module, the low concentration group, concentration and high concentration groups, determined by MTT method was applied to explore the above categories in cell proliferation, cell apoptosis was measured by flow cytometry, the expression of inflammatory factors in cell supernatant was determined by enzyme-linked immunoassay, and the expression of autophagy marker proteins LC3- ? and P62 was determined by Western-Bolt method. In order to explore the relationship between AMPK and SIRT1, immunoco-precipitation method was used to determine the interaction between AMPK and SIRT1, and dual luciferase experiment was used to explore the effect of AMPK on SIRT1. The AICAR group, BLM-275 group and negative control group were established. To explore the effect of SIRT1 on AMPK, we established SRT 1720 group, EX-527 group and control group. Direct binding between AMPK and SIRT1 should be determined by chromatin co-precipitation assay. In order to further explore the effect of AMPK/SIRT1 positive feedback loop on the systemic inflammatory response of acute pancreatitis, this study selected the medium-concentration Danggui Shaoyajiao SAN group as the control group (group C), and applied AMPK inhibitor BLM-275 and SIRT1 inhibitor EX 527 to the effect of medium-concentration Danggui Shaoyajiao SAN cells, respectively. The expression of autophagy marker proteins LC3- ? and P62 in groups A and B were determined by the Western-Bolt method. Results showed that compared with the control group, the cell survival rate, the expression of AMPK, SIRT1 and LC3-II in the model group were decreased, and the apoptosis rate of iNOS, IL-2, TNF-?, P62 and apoptosis were increased in the model group (P<0.05). the levels of iNOS, IL-2, TNF-?, P62 and cell survival rate in low, medium and high concentration groups decreased gradually, while the expressions of AMPK, SIRT1, LC3-II and cell apoptosis rate increased (P<0.05). The levels of iNOS, IL-2 and TNF-? in the three groups were gradually decreased with the increase of the concentration (P<0.05). Immunoprecipitation showed that AMPK and SIRT1 could bind to each other in cells. The double luciferase experiment indicated that the reporter gene containing the SIRT1 binding site was constructed. The luciferase activity was increased in THE AICAR group and decreased in the BLM-275 group (P<0.05). The reporter gene containing the AMPK promoter binding site was constructed. The luciferase activity in SRT1720 group was increased, while that in EX-527 group was decreased. SIRT1 could directly bind to the AMPK promoter. SIRT1 and LC3- ? protein expressions in group A were down-regulated, and P62 protein was increased (P<0.05). The protein expressions of AMPK and LC3- ? in group B were down-regulated, and the protein expression of P62 was increased (P<0.05). It concluded that AMPK can directly bind to activate SIRT1 expression, and SIRT1 expression can also activate AMPK, forming a positive feedback loop between the two. Therefore, Angelic Shaoyaodong decoction can mediate AMPK/SIRT1 positive feedback pathway to promote autophagy and regulate systemic inflammatory response in acute pancreatitis.

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151 A NOVEL METHOD TO INCREASE THE DEVELOPMENTAL POTENTIAL OF ACTIVATED OOCYTES BY USING THE Zn2+ CHELATOR TPEN [N,N,N',N'-TETRAKIS(2-PYRIDYLMETHYL)ETHYLENEDIAMINE]

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab151 ◽

2014 ◽

Vol 26 (1) ◽

pp. 189

Author(s):

K. Lee ◽

A. Davis ◽

C. N. Murphy ◽

R. S. Prather

Keyword(s):

Positive Feedback ◽

Feedback Loop ◽

Cell Number ◽

Total Cell ◽

Control Group ◽

Oocyte Activation ◽

Blastocyst Formation ◽

High Concentration ◽

Positive Feedback Loop ◽

Developmental Potential

An increase in intracellular Ca2+ concentration is essential for oocyte activation. Thus most artificial oocyte-activation methods focus on increasing the Ca2+ concentration in the oocytes. Recently, full-term development was reported in mice when oocytes were activated with no increase in intracellular Ca2+ using the Zn2+ chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). During oocyte maturation, Zn2+ is responsible for Cdc25c recognition. Once attached, the Cdc25c-zinc complex dephosphorylates maturation-promoting factor (MPF)/cyclin-dependent kinase 1 (cdk1), thus activating the Cdc25c-MPF positive feedback loop and keeping the oocyte suspended in metaphase II. The TPEN can inhibit the Cdc25c-MPF positive feedback loop, indirectly giving TPEN the power to degrade MPF, allowing the oocyte to exit metaphase II. First, we tested if incubation of porcine oocytes with TPEN could induce oocyte activation. Second, we examined whether the combination of TPEN with conventional activation methods could increase the developmental potential of activated oocytes. Last, based on the results, somatic cell nuclear transfer (SCNT) embryos were further activated with the optimum condition of TPEN to produce clones to confirm developmental competence. Frequencies of blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell numbers in blastocysts were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. When oocytes were incubated with a high concentration of TPEN (100–250 μM) for 10 to 120 min, blastocyst formation was comparable with conventional activation methods; however, the total cell number in the blastocysts was significantly lower (31.3 ± 3.1 v. 24.8 ± 1.9). When oocytes were activated with conventional methods and then incubated with the high concentration of TPEN, embryo development was drastically decreased; no blastocyst development was achieved from TPEN-treated oocytes. Interestingly, when activated oocytes were incubated with a low concentration of TPEN (5–10 μM), surprisingly, the TPEN-treated group showed higher developmental potential compared with the control group. Specifically, the average percent blastocyst formation of TPEN-treated oocytes (5 μM for 30 min) was 27.2 ± 1.7%, but only 10.6 ± 2.5% developed to blastocyst in the control group. Moreover, the average cell number in blastocysts was significantly higher in TPEN-treated oocytes compared with the control group (33.1 ± 2.6 v. 28.2 ± 2.1, respectively). When 290 chemically activated SCNT embryos were treated with 5 μM TPEN for 30 min and transferred into a surrogate, 2 healthy piglets were born. The results indicate that incubation of oocytes with TPEN alone can activate porcine oocytes. Also, when activated oocytes are incubated with the right concentration of TPEN, it can increase embryo quality in vitro. Embryo transfer results also show that TPEN-incubated SCNT embryos are developmentally competent. Additional studies would guide us to develop more efficient way to use TPEN in the activation of SCNT embryos.

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Effects of Micro-Ecological Enteral Nutrition on Systemic Inflammatory Response, Bacterial Translocation, and Immune Function in Patients with Severe Acute Pancreatitis

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.19:240-247 ◽

2020 ◽

Vol 19 (3) ◽

pp. 240-247

Author(s):

Yilan Wang ◽

Hongwei Ye ◽

Feng Zheng ◽

Xinsen Zou ◽

Xianbin Wu ◽

...

Keyword(s):

Acute Pancreatitis ◽

Enteral Nutrition ◽

Inflammatory Response ◽

Immune Function ◽

Severe Acute Pancreatitis ◽

Bacterial Translocation ◽

Severity Index ◽

Systemic Inflammatory Response ◽

Mucosal Barrier ◽

Control Group

We have evaluated the effect of early and late micro-ecological enteral nutrition on systemic inflammatory response, bacterial translocation, and immune function in patients with severe acute pancreatitis. Two weeks after treatment, experimental group receiving early nutritional intervention exhibited a significant increase in intestinal lactobacilli and bifidobacteria, fewer staphylococci, and E. coli, and lower levels of serum endotoxin, D-lactic acid, and diamine oxidase than the group receiving late intervention (control group) (P < 0.05). Also, the serum levels of CD3+ and CD4+ and CD4+/CD8+ ratio significantly increase after 2 weeks of intervention. On the other hand, the level of CD8+ and the Acute Physiology and Chronic Health Evaluation II and Modified Computed Tomography Severity Index scores significantly decreased after 2 weeks of treatment (P < 0.05). The early intervention also led to a significant shortening of time needed for abdominal pain relief, anal exhaustion, resumption of peristaltic sound, and hospitalization. Furthermore, there was a significant increase in overall response rate, and decrease in the incidence rate of complications (P < 0.05). Early micro-ecological enteral nutrition therapy can effectively relieve systemic inflammatory response, prevent intestinal bacterial translocation, restore the function of intestinal mucosal barrier, and alleviate nutritional imbalance in severe acute pancreatitis patients leading to improved immune function, mitigation of the disease, and reduced complications benefiting the recovery of patients.

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