MOTS-c mimics exercise to combat diabetic liver fibrosis by targeting Keap1-Nrf2-Smad2/3

Abstract

Liver fibrosis is a common complication of T2DM(Type 2 diabetes mellitus). Appropriate intervention (exercise or drugs) in the early stage of liver fibrosis can slow down or even reverse liver fibrosis. MOTS-c (Mitochondrial open reading frame of the 12 S r RNA type-c ) has been described as an exercise-mimicking substance, and its effects are similar to those achieved by aerobic exercise; however, the exact mechanism remains to be elucidated. In this study, liver function was impaired in a T2DM rat model, leading to the aggravation of liver fibrosis. T2DM rats with liver fibrosis were subjected to MOTS-c, aerobic exercise therapy, or their combination. HE staining, Masson's trichrome staining and immunohistochemistry were used for histopathological examination. Transcriptome sequencing, q-PCR and WB were used to detect the expression of Keap1 (Kelch-like ECH-associated protein 1), Nrf2 (Nuclear factor erythroid 2-related factor 2 ), Smad2/3/4 and other genes. MOTS-c and aerobic exercise therapy improved T2DM-induced liver fibrosis. Additionally, cells were transfected with MOTS-c overexpression or interference plasmids or MOTS-c was added to the culture medium. MOTS-c overexpression or MOTS-c addition to the culture medium inhibited ROS levels, increased the mRNA and protein expression of Keap1-Nrf2 pathway genes and decreased the expression of TGF-β1(Transforming growth factor-beta1)/Smad pathway genes. Our findings demonstrate that MOTS-c modulates the progression of T2DM complicated by liver fibrosis through a Keap1-Nrf2-Smad2/3 signaling pathway-dependent mechanism.

Keywords: Aerobic exercise; Keap1-Nrf2-Smad2/3 pathway; Liver fibrosis; MOTS-c; T2DM.

Fig. 1

MOTS-c and aerobic exercise alleviated liver tissue damage caused by T2DM. (A) Analytical balance was used to measure the mean body weight of rats in control group (C), diabetic group (D), diabetic exercise group (DE), diabetic MOTS-c group (DM), and diabetic exercise + MOTS-c group (DME). Following confirmation of successful diabetic model establishment after a 7-week high-fat/high-sucrose dietary induction period, longitudinal body weight measurements were conducted weekly throughout the subsequent 8-week experimental phase. (B) Fasting insulin was measured in each group.(C)Blood samples (0.05 mL) were collected from the tail vein of each group to measure fasting blood glucose (FBG). (D) Homeostasis model assessment of insulin resistance (HOMA-IR) was performed as follows: [Plasma glucose (GLU, mmol/L) X serum insulin (m IU/L)]/22.5. (E) Blood was collected from the five groups and tested according to the procedure of the HbA1c detection kit. ALT(F) and AST (G) in the five groups were measured using ELISA kits. HE (H) and Masson (I) staining were used to detect the pathological changes of liver tissues in the 5 groups (C, D, DE, DM and DME group). All results are given as the mean ± standard deviation (SD). n = 3, * p < 0.05, ** p < 0.01.

Fig. 2

MOTS-C and aerobic exercise modulate liver fibrosis in T2DM rats through the Keap1-Nrf2-Smad2/3 pathway. By transcriptome sequencing, the differentially expressed genes in the 5 groups (C, D, DE, DM and DME group) were analyzed by bar chart (A) and Venn diagram(B). (C) Based on transcriptome data, the expression of Nrf2, Keap1, Gclm, Gclm, Ho-1, Tgf-β1, Smad2/3/4, Col I, Col III and α-Sma in 5 groups were analyzed by cluster heat map. (D) Real-time qPCR was used to quantify the mRNA expression levels of Nrf2, Keap1, Gclm, Gclm, Ho-1, Tgf-β1, Smad2/3/4, Col I, Col III and α-Sma in the liver tissue of the five groups. (E) Western blotting was employed to determine the protein abundances of MOTS-c, NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III, and α-SMA in the liver tissue of the five groups. The intensity of those immunoblots, representing different protein expression levels, was also quantified by the Quantity One 4.6.2 software, as shown in the figure. The quantitative data was reflected in the numbers above each protein. The larger the number, the higher the gray level.The WB experiment results in the paper came from the same complete gel, but it was cut in the nurturing process to separate the nurturing into different proteins. All results are given as the mean ± standard deviation (SD). n = 3, ** p < 0.01.

Fig. 3

The expression of intracellular antigen in liver tissue of 5 groups was detected by immunohistochemical method. Immunohistochemistry was used to detect the expression of Nrf2 (A), Keap1 (B), TGF-β1 (C), Smad2 (D), Col I (E), Col III (F) and α-Sma (G) in the liver tissue of the five groups, and the results were analyzed by Histochemistry score (H-score). The area pointed by the arrow was the main positive area.n = 3, ** p < 0.01.

Fig. 4

Both endogenous and exogenous MOTS-c inhibited ROS in LX-2, LO-2 and HepG2 cells. (A) We transfected three different MOTS-c interfering RNA (si-RNA-1, si-RNA-2 and si-RNA-3) into LX-2, LO-2 and HeapG2 cells, respectively. The protein abundance of MOTS-c was visualized by Western blotting with indicated antibodies. (B) The MOTS-c overexpression plasmid was transfected into LX-2, LO-2 and HeapG2 cells, respectively. Western blotting was employed to determine the protein abundances of MOTS-c. (C) After transfection of pcDNA3.1, MOTS-c and si-RNA-3 into cells, the three treatments were named NC group, MOTS-c group and si-RNA-3 group, respectively. ROS was determined by flow cytometry. (D) Exogenous MOTS-c was supplemented into the complete culture medium for the cultivation of three distinct cells Cells were divided into two groups according to whether exogenous MOTS-c was added or not: NC group and MOTS-c group (10 µM,24 h). ROS was determined by flow cytometry. The intensity of those immunoblots, representing different protein expression levels, was also quantified by the Quantity One 4.6.2 software, as shown in the figure. The quantitative data was reflected in the numbers above each protein. The larger the number, the higher the gray level.The results were also graphically shown in (C-D), with statistical significances determined by using MANOVA.The WB experiment results in the paper came from the same complete gel, but it was cut in the nurturing process to separate the nurturing into different proteins. n = 3, * p < 0.05,** p < 0.01.

Fig. 5

Endogenous MOTS-c inhibited the expression of TGF-β1-Smads pathway through Nrf2 in LX-2, LO-2 and HepG2 cells. After transfection of pcDNA3.1, MOTS-c and si-RNA-3 into cells, the three treatments were named NC group, MOTS-c group and si-RNA-3 group, respectively. (A) Real-time qPCR was used to quantify the mRNA expression levels of NRF2, KEAP1, GCLC, GCLM, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LX-2 cell lines. (B) Western blotting was employed to determine the protein abundances of NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LX-2 cell lines. (C) The mRNA expression levels of NRF2, KEAP1, GCLC, GCLM, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LO-2 cell lines were determined by real-time qPCR. (D) The protein abundances of NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LO-2 cell lines were visualized by Western blotting with indicated antibodies. (E) Real-time qPCR was used to quantify the mRNA expression levels of NRF2, KEAP1, GCLC, GCLM, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in HepG2 cell lines. (F) Western blotting was employed to determine the protein abundances of NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in HepG2 cell lines. The intensity of those immunoblots, representing different protein expression levels, was also quantified by the Quantity One 4.6.2 software, as shown in the figure. The quantitative data was reflected in the numbers above each protein. The larger the number, the higher the gray level. The WB experiment results in the paper came from the same complete gel, but it was cut in the nurturing process to separate the nurturing into different proteins. All results are given as the mean ± SD. n = 3, ** p < 0.01.

Fig. 6

Exogenous MOTS-c inhibited the expression of TGF-β1-Smads pathway through Nrf2 in LX-2, LO-2 and HepG2 cells. Exogenous MOTS-c was supplemented into the complete culture medium for the cultivation of three distinct cells.Cells were divided into two groups according to whether exogenous MOTS-c was added or not: NC group and MOTS-c group (10 µM,24 h). (A) Real-time qPCR was used to quantify the mRNA expression levels of NRF2, KEAP1, GCLC, GCLM, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LX-2 cell lines. (B) Western blotting was employed to determine the protein abundances of NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LX-2 cell lines. (C) The mRNA expression levels of NRF2, KEAP1, GCLC, GCLM, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LO-2 cell lines were determined by real-time qPCR. (D) The protein abundances of NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in LO-2 cell lines were visualized by Western blotting with indicated antibodies. (E) Real-time qPCR was used to quantify the mRNA expression levels of NRF2, KEAP1, GCLC, GCLM, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in HepG2 cell lines. (F) Western blotting was employed to determine the protein abundances of NRF2, KEAP1, GCLC, HO-1, TGF-β1, SMAD2/3/4, COL I, COL III and α-SMA in HepG2 cell lines. The intensity of those immunoblots, representing different protein expression levels, was also quantified by the Quantity One 4.6.2 software, as shown in the figure. The quantitative data was reflected in the numbers above each protein. The larger the number, the higher the gray level.The WB experiment results in the paper came from the same complete gel, but it was cut in the nurturing process to separate the nurturing into different proteins. All results are given as the mean ± SD. n = 3, ** p < 0.01.