Verapamil induces autophagy to improve liver regeneration in non-alcoholic fatty liver mice.

Authors: Jian-Lin Lai , Yuan-E Lian , Jun-Yi Wu , Yao-Dong Wang , Yan-Nan Bai ,
Shengli Clinical Medical College of Fujian Medical University, Department of Hepatobiliary and Pancreatic Surgery, Fujian Provincial Hospital, Fuzhou, Fujian 350001, China.
Co-corresponding author: Yao-Dong Wang , Yan-Nan Bai .
E-mail address: 42236640@qq.com.

Generation Introduction:
To sum up, we proved that autophagy plays a key role in promoting liver regeneration, alleviating liver injury, and prolonging mice's survival after PH. The pharmacological regulation of verapamil on autophagy effectively promoted liver regeneration, hepatocyte production, and survival in mice and reduced liver injury in mice after PH. Autophagy is expected to become a key target for NAFLD treatment and open up a new direction for the prevention and treatment of obesity, T2DM, Mets, and other diseases.

The data in this data set was collected in the Fujian Medical University between May 2019 and March 2021.


Description of the data in this data set:
Fig. 1. The mice with different degrees of fatty liver were successfully modeled. A: The general morphology of liver in the control group, mild to the moderately fatty liver group, severe fatty liver group, and steatohepatitis group; B: The degree of steatosis was observed by HE staining in four groups. C: Four groups of liver parenchyma Oil Red O staining; D. The hepatocytes' ultrastructure was observed by TEM; E:Four groups of biochemical indexes. (*:0.01<P<0.05, **: 0.001<P<0.01, ***: 0.0001<P<0.001,****: P<0.0001, n=10, HE and Red O staining scale bar: 100m.)

Fig. 2. Autophagy was observed in the control group (ND4m), mild to moderately fatty liver group (HFD2m), severe fatty liver group (HFD4m), and fatty hepatitis group (HFD+CCl4) at rest and after hepatectomy. A: The autophagy state of the four groups in the resting state, WB detection of autophagy-related proteins LC3II/I, p62, Beclin1, Atg7; B: WB to detect the changing trend of LC3II/I of the four groups; C: WB detection of p62; D: WB detection of Beclin1; E: WB detection of Atg7; F: LC3 and LAMP1 immunofluorescence co-localization imaging of the ND4m and HFD2m after PH 48 h; G: LC3 and LAMP1 immunofluorescence co-localization imaging of the HFD4m and HFD+CCl4 after PH 72 h. The yellow arrows represented autophagolysosomes, scale bar: 40m.

Fig. 3. After 50% hepatectomy, liver regeneration was performed in the ND4m, HFD2m, HFD4m, and HFD+CCl4. A: The changing trend of liver regeneration rate in the four groups at 0, 6, 12, 24, 48, 72, 120, and 168 h after hepatectomy; B: The expression of BrdU which was detected by the immunohistochemical method after PH; C: The expression of PCNA after PH. Scale bar: 20m.

Fig. 4. The changes in body weight, liver morphology, liver function, oxidative stress, and autophagy of mice in the HFD2m and HFD4m after intraperitoneal verapamil injection. A: After injection of verapamil 25 mg/kg for 10 d, the changes of general morphology, histological status, and oil red staining; B: The changes of body weight within 10 d after injection of verapamil; C: The changes of GLU, AST and AST in the HFD2m after verapamil injection; D: The changes of the HFD4m's GLU, AST, and AST after verapamil injection; E: The changes of SOD, GSH, MDA, and POD after verapamil injection; F: Changes of autophagy-associated proteins in mice injected with verapamil; G, H: LC3 and LAMP1 IF co-localization imaging, the yellow arrows represented autophagolysosomes. *: 0.01<P<0.05, **: 0.001<P<0.01, ***: 0.0001<P<0.001, ****: P<0.0001, n=10, HE and Red Ostaining scale bar: 100m, IF scale bar: 40m.

Fig. 5. Autophagy changes of mice in HFD2m and HFD4m after 50% hepatectomy after injection of verapamil. A: Comparison of autophagy levels at 24, 48, 72, and 120 h after hepatectomy in HFD2m after injection of normal saline and verapamil respectively; B: Comparison of autophagy level after hepatectomy in HFD4m after injection of normal saline and verapamil; C, D: LC3 and LAMP1 IF co-localization imaging (48 h after hepatectomy in the HFD2m and 72 h after hepatectomy in the HFD4m, the yellow arrows represented autophagolysosomes, scale bar: 40m); E: Obvious autophagosomes were observed after hepatectomy, and the yellow arrows represented autophagosomes.

Fig. 6. The HFD2m and HFD4m were injected with normal saline and verapamil, respectively, after hepatectomy. A: The changes of liver weight to body weight ratio at different time points after hepatectomy in the two groups after injection of normal saline and verapamil respectively; B: After injection of normal saline and verapamil, the expression of BrdU which was detected by the immunohistochemical method after PH; C: The expression of PCNA after PH; D: Quantitative results of BrdU and PCNA. (*: 0.01<P<0.05, **: 0.001<P<0.01, ***: 0.0001<P<0.001, ****: P<0.0001, n=10, scale bar: 20m.)

Fig. 7. Detection of mTOR signal pathway protein after verapamil injection. A: The expression of the mTOR signal pathway protein in liver tissue of mice with mild to a moderately fatty liver after PH; B: Expression of the severe fatty liver's mTOR signal pathway protein after PH.

Supplementary Fig. 1. Autophagy-related genes (LC3/p62/Beclin1/Atg7) mRNA levels were measured by RT-qPCR in corresponding liver samples in the control group (ND4m), mild to moderately fatty liver group (HFD2m), severe fatty liver group (HFD4m), and fatty hepatitis group (HFD+CCl4) at rest and after hepatectomy. A: The mRNA levels of LC3/p62/Beclin1/Atg7 at resting state; B: q-PCR to detect the changing trend of LC3 mRNA level after hepatectomy; C: q-PCR to detect the changing trend of p62 mRNA level after hepatectomy; D: q-PCR to detect the changing trend of Beclin1 mRNA level after hepatectomy; E: q-PCR to detect the changing trend of Atg7 mRNA level after hepatectomy. (*: 0.01<P<0.05, **: 0.001<P<0.01, ***: 0.0001<P<0.001, ****: P<0.0001, n=10.)

Supplementary Fig. 2. The protein levels of mTOR, p-mTOR, 4EBP1, p-4EBP1, P70S6K, p-P70S6K in total liver after VER rejection and hepatectomy were assessed by western blot, quantified using ImageJ analysis and normalized to GAPDH. A: The relative intensity of p-mTOR/mTOR protein in liver tissue of mice with mild to a moderately fatty liver; B: The relative intensity of p-4EBP1/4EBP1 protein in liver tissue of mice with mild to a moderately fatty liver; C: The relative intensity of p-P70S6K/P70S6K protein in liver tissue of mice with mild to a moderately fatty liver; D: The relative intensity of p-mTOR/mTOR protein in liver tissue of mice with severe fatty liver; E: The relative intensity of p-4EBP1/4EBP1 protein in liver tissue of mice with severe fatty liver; F: The relative intensity of p-P70S6K/P70S6K protein in liver tissue of mice with severe fatty liver.

Materials and methods:
Grouping of experimental animals and animal models
Male C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Company. After one week of adaptive feeding, 5-week-old healthy mice were randomly divided into normal group, mild to the moderately fatty liver group, severe fatty liver group, and steatohepatitis group. All the animals were raised in the Animal Experimental Center of Fujian Medical University. The laboratory's constant temperature was 22±1℃, the relative humidity was 55±5%, and the cycle of day and night was 12 h. Before hepatectomy, there was no food or drink for 12 h. All the experiments were carried out following the ethical review requirements of animal experiments at Fujian Medical University.
Mice in the control group were fed with D12450B forage for 4 months. The fatty liver groups were fed with a high-fat diet D12490 for different times, the mild to the moderately fatty liver group was fed for 2 months, and the severe fatty liver group was fed for 4 months. After 4 months, the steatohepatitis group was intraperitoneally injected with 20% carbon tetrachloride (CCl4, 0.5ml per kg body weight) twice a week for 2 weeks. Finally, the model's success was confirmed by mouse body weight, liver tissue HE staining, and oil red O staining, according to the range of hepatocyte steatosis. Through semi-quantitative assessment of the degree of steatosis, 5%-33% were mild steatosis, 33%-66% were moderate steatosis, and more than 66% were severe steatosis. Ballooning degeneration and intralobular inflammation of the liver were steatohepatitis. They were divided into a normal group (ND4m), mild to moderately fatty liver group (HFD2m), severe fatty liver group (HFD4m), and fatty hepatitis group (HFD+CCl4). Then, the mild to moderately fatty liver andsevere fatty liver groups were intraperitoneally injected with verapamil (25 mg per kg body-weight for 10 d), while the control group was injected with the same dose of normal saline, which was divided into HFD2m+NS, HFD2m+VER, HFD4m+NS, and HFD4m+VER.

50% hepatectomy model(Partial hepatectomy, PH)
After establishing the fatty liver model, 50% hepatectomy was performed to establish the liver regeneration model. Surgery was performed under 2% isoflurane continuous inhalation anesthesia. According to Higgins's hepatectomy, the left lateral and right lobes (right upper lobe and right lower lobe) of the liver were resected.The mice were killed at 0, 12, 24, 36, 48, 72, 120, and 168h after hepatectomy, blood was collected and centrifuged, and plasma stored at -80 ℃. Livers were removed, weighed, and thin slices of all livers lobes immersed in 10 percent formalin or snap-frozen immediately in liquid nitrogen for further analysis.

Detection of liver function and liver metabolic indexes
The body-weight of mice was detected by electronic balance before an operation or before drug injection in the morning. The detection of biochemical blood indexes showed that the amount of blood collected by eyeball puncture or eyeball extraction was about 0.5-1.0ml before the operation, and at each time point after an operation, the blood was kept on ice for 30 min, and the upper serum was kept at -80℃ after 10min centrifuge at 8000 rpm/min with low temperature (-4℃). Glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were detected by rate method on an automatic biochemical analyzer. The oxidative stress reactive oxygen species (ROS) index was detected when the liver tissue was cut off at different time points after an operation and preserved at -80℃. During the detection, the liver tissue homogenate was prepared according to the instructions, and then malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and GLUtathione (GSH) were detected according to the ROS detection kit manual.

Detection of liver regeneration indexes
The resected and residual livers' weight was weighed at 0 (operation), 6, 12, 24, 48, 72, 120, and 168 h after PH, respectively.The liver's coefficient to body weight was calculated as the ratio of wet liver weight to body weight. Liver regeneration rate was expressed as a percentage of regenerated liver mass calculated using the equation [C-(A-B)]/A, where A was the estimated total liver mass at the time of liver resection, B was the resected liver's wet weight,and C was the residual liver mass regenerated at the corresponding time. Proliferating cell nuclear antigen (PCNA) expression was also used to evaluate liver, according to the immunohistochemical staining steps to detect hepatocyte PCNA quantify. One hour prior to sacrifice at different time points after PH, a single injection of 10mg/ml BrdU (5-bromo-2'-deoxyuridine) was administered intraperitoneally at a dose of 100 mg/kg animal weight. Liver tissue BrdU was detected by immunohistochemistry and quantified by the same method as above.

Detection of autophagy index
Western blotting (WB): after hepatectomy, the liver tissue was stored at -80℃. The regenerated liver tissue of each group was ground with liquid nitrogen.The liver tissue was dissociated with proteolysis solution, the total protein of liver tissue was extracted.The protein content was determined. The supernatant was collected and added to the sample buffer, boiled in boiling water at 100℃ for 5 min, and stored at -20℃. Western blotting was used to detect the markers of autophagy-related proteins, such as LC3, p62, Beclin1, Atg7, mTOR, etc.
Immunofluorescence (IF): after hepatectomy, the liver tissue was stored at -80℃ and transported at low temperature. After paraffin embedding, frozen sections were made and stored at -20℃. According to immunofluorescence imaging, the co-localization of LC3 and LAMP1 was observed to determine the binding of autophagosomes and lysosomes.

Examination by transmission electron microscope (TEM)
For TEM, liver biopsies were fixed in glutaraldehyde(3%) plus p-formaldehyde (1.5%) for several hours. Samples were postfixed in 1% osmium tetroxide for 90 min at 25 ℃, stained with uranyl acetate (5 mg/ml) for 1.5 h at 25 ℃, dehydrated in acetone and embedded. Ultra-thin sections, unstained or poststained with uranyl acetate and lead hydroxide, were examined under a TECNAI transmission electron microscope of FEI Company. The cellular structure, autophagosomes, and organelles such as mitochondria and lysosomes in the process of liver regeneration were observed.

Main reagents
High-fat diet D12490 and control diet D12450B were purchased from the American Diet-Research Company. Saturated Oil Red O dyeing solution was obtained from Beijing Solarbio Technology Co., Ltd. Malondialdehyde (MDA) kit A003-1, superoxide dismutase (SOD) kit A001-3, peroxidase (POD) kit A084-1, glutathione (GSH) test kit A006-1 were from Nanjing Jiancheng Institute of Biological Engineering.Verapamil hydrochloride (V4629) was purchased from Sigma-Aldrich of the United States. The membranes were immunoblotted with anti-LC3 (Abcam, ab19, Cambridge, UK), anti-Proliferating Cell Nuclear Antigen (PCNA) (Abcam, ab29, Cambridge, UK), anti-Beclin 1 (Abcam, ab207612, Cambridge, UK), anti-p62/SQSTM1 (Santa Cruz Biotechnology, Sc-28359, Santa Cruz, CA, USA), anti-Atg7, anti-phosphorylated and total mTOR, anti-phosphorylated and total 4EBP1, anti-phosphorylated and total p70 S6 Kinase (Cell Signaling Technology, #2631, #4060, #9272, #2971, #2983, #9205, #2708, Beverly, MA, USA). Carbon tetrachloride (CCl4) and olive oil were from Shanghai Xinzhong Chemical Reagent Factory.