SHYCD induces APE1/Ref-1 subcellular localization to regulate the p53-apoptosis signaling pathway in the prevention and treatment of acute on chronic liver failure

Abstract

Background & Aims: San huang yin chi decoction(SHYCD) is derived from the yin chen hao decoction, a well-known and canonical Chinese medicine formula from the "Treatise on Febrile Diseases". Over the past decade, SHYCD has been used to treat and prevent the liver cirrhosis and liver failure. In the present study, we investigated the effects of SHYCD for acute on chronic liver failure(ACLF) and explored its potential mechanism. an ACLF rat model, which induced by carbon tetrachloride (CCl4) combined with D-galactosamine (D-GalN) and lipopolysaccharide(LPS), was used and confirmed by B-ultrasound analysis. Rats were randomly divided into control group, model group, SHYCD-H group, SHYCD-M group, SHYCD-L group, AGNHW group. Compared with the ACLF model group, High, medium, and low doses of SHYCD reduced ALT, AST, TBIL, NH3, IL-1β, IL-6, and TNFα expression levels in the serum, Shorten PT and INR time,and increased Fbg content in the whole blood, increased survival rate of the rats, improved liver pathological changes. APE1 / Ref-1 was mainly expressed in the nucleus, but the nucleus and cytoplasm were co-expressed after hepatocyte injury. SHYCD significantly downregulated APE1/Ref-1 expression in the cytoplasm. Increased APE1/Ref-1, Bcl-2, reduced p53, caspase-3, Bax, and Cyt-c in the total protein. Base on the results, we conclused that High, medium, and low doses of SHYCD could be applied in prevention and treatment of ACLF, and dose-dependent. The possible mechanism is to promote the APE1 / Ref-1 from the cytoplasm to the nuclear transfer, regulation of p53 apoptosis signal pathway prevention and treatment of ACLF.

Keywords: APE1/Ref-1; Pathology Section; San huang yin chi decoction; acute on chronic liver failure; apoptosis; p53.

Figure 1. Modeling effects in rats and SHYCD effects on rat survival state

a. B -Ultrasound imaging results for rat liver, spleen size, and number of abdominal ascites. b. Macroscopic observation of the liver gloss, color, form, and number of abdominal ascites. c., d. Comparison between the ACLF model group and control group showing that liver and spleen weight ratio to body weight increased significantly in ACLF model group (**P < 0.01; *P < 0.05, vs. model). e. Observation of ACLF rat model group survival curves within 48 hours based on carbon tetrachloride toxicity and joint D -GalN and LPS acute attack mimicking ACLF.

Figure 2. The influence of SHYCD on rat liver function, blood coagulation, and blood ammonia levels

a. Values for ALT, AST, and TBIL as the three indicators of liver function. b. Values for PT, INR, and Fbg as the three indicators of coagulant function. c. Blood ammonia level. *p < 0.05; **p < 0.01 VS model.

Figure 3. The effects of SHYCD for ACLF on the Histological and Ultrastructural changes in the liver tissue

a. Representative microscopic images of HE stain. 200x b. TEM images of ultrathin sections of myocardial tissue are changed,15000x. A: control group;B:Model group; C:SHYCD-H group; D: SHYCD-M group; E: SHYCD-L group; F:AGNHW group.

Figure 4. The effects of SHYCD on APE1 mRNA and protein levels

a. Effect of SHYCD on APE1 mRNA b. Confocal immunofluorescence microscopy image of APE1 in situ hybridization (400x). A: control group; B: model group; C:SHYCD-H group; D: SHYCD-M group; E: SHYCD-L group; F:AGNHW group,*p < 0.05; **p < 0.01,VS model. c. Western blot of APE expression in liver tissue nucleoprotein and cytoplasm protein. N-pro: nucleoprotein, C-pro: cytoplasm protein, T-pro:total protein. *p < 0.05; **p < 0.01,VS model.

Figure 5. APE1 interaction with P53

a. Confocal immunofluorescence microscopy image of p53 in situ hybridization (400x). A: control group; B: model group; C:SHYCD-H group; D: SHYCD-M group; E: SHYCD-L group; F:AGNHW group,*p < 0.05; **p < 0.01,VS model. b. Western blot analysis of p53 expression,*p < 0.05; **p < 0.01,VS model. (c) IP performed with an anti-APE1 antibody. Co-IP P53 and APE1 were detected by Western blot.

Figure 6. SHYCD reduced Bax, cleaved caspase-3, and Cyt-C, and increased BCL-2

SHYCD also inhibited hepatocyte apoptosis a. Western blot results showing that SHYCD reduced Bax, cleaved caspase-3, and Cyt-C, and increased BCL-2. b. Immunohistochemistry results showing that SHYCD reduced Bax, cleaved caspase-3, and Cyt-C, and increased BCL-2 (200x). c. Tunel results showing that SHYCD reduced hepatocyte apoptosis (200x). *p < 0.05; **p < 0.01, VS model. A: control group; B: model group; C:SHYCD-H group; D: SHYCD-M group; E: SHYCD-L group; F:AGNHW group.

Figure 7. current working model