The effect and mechanism of SHP2 on sepsis induced myocardial injury via modulating Src/ERK-autophagy signal

Mouse ultrasound electrocardiogram results

The results of echocardiography showed that compared with the sham group, the LVEF, LVFS and E/A of the mice in the model group and PHPS1 group were significantly decreased, while the LVEF, LVFS and E/A of the mice in the PHPS1 group were significantly decreased compared with the model group (Figure 1). The results showed that SHP2 could significantly improve cardiac dysfunction caused by myocardial injury in sepsis.

Figure 1. Ultrasound electrocardiogram examination results of mice in each group. (A) Left ventricular echocardiography of mice; (B) Statistical results of LVEF, LVFS, E/A ratios of left ventricle in mice, data was presented as X±S. Comparisons between sham group and model group, **p<0.01; Comparisons between sham group and PHPS1 group, **p<0.01; Comparisons between model group and PHPS1 group, **p<0.01.

Results of HE staining in mice myocardium

The results of HE staining showed that the myocardial cells of the sham group were arranged orderly, and no pathological damage such as necrosis, hyperemia and edema was found. In the model group, the arrangement of myocardial cells was disordered, with rupture of sarcolemmal membrane and enlargement of nucleus, accompanied by slight necrosis, interstitial hyperemia, edema and infiltration of inflammatory cells. Compared with the model group, the arrangement of cardiomyocytes in the PHPS1 group was disordered, and the conditions of nuclear necrosis, interstitial hyperemia and inflammatory cell infiltration were significantly worse (Figure 2), turning out that SHP2 can ameliorate myocardial pathology caused by myocardial injury caused by sepsis.

Figure 2. HE staining results of myocardium in mice of each group.

Results of transmission electron microscopy of mouse cardiomyocytes

The results of transmission electron microscope indicated that the myocardial fibers of mice in sham group were arranged in order, the mitochondrial cristae was tight, the mitochondrial double membrane was not damaged, without dissolution, vacuole and other phenomena, the autophagy phenomenon was weak, and the number of lysosomes was less; In model group, the myocardial fibers were broken, the sarcomere was arranged disorderly, some mitochondria were swollen and broken, vacuolated, the ridge structure was disordered, and the number of autophagy bodies and lysosomes increased. In the PHPS1 group, severe myocardial fiber rupture was severe, sarcomere arrangement disorder was aggregated, a large number of mitochondria swelling and rupture was present, vacuolation, ridge structure was disorder, autophagy body and lysosome increased significantly (Figure 3). It indicated that SHP2 could mitigate the ultrastructural changes of myocardial cells induced by sepsis.

Figure 3. Transmission electron microscopic examination of myocardial cells in each group.

Results of the detection of p-SHP2, p-Src and p-ERK proteins in mice cardiomyocytes

The western blot results showed that the relative protein expressions of p-Src and p-ERK1/2 in the sham operation group were significantly lower than those in the model group and PHPS1 group, while the relative protein expressions of p-Src and p-ERK1/2 in the PHPS1 group were significantly higher than those in the model group. Compared with the sham operation group, the relative protein expression of p-SHP2 in the model group was significantly increased, and the relative protein expression of p-SHP2 in the PHPS1 group was significantly decreased (Figure 4). These results showed that SHP2 could suppress the protein activity of Src and ERK in myocardial injury induced by sepsis.

Figure 4. Western blot results of p-SHP2, p-Src and p-ERK protein in myocardial cells of mice in each group. (A) Western blot results of p-SHP2, p-Src and p-ERK1/2 in mouse cardiomyocytes in each group; (B) Statistical diagram of the expression levels of p-SHP2, p-Src and p-ERK1/2 proteins in mouse cardiomyocytes in each group, and the data was shown as X±S. Comparisons between sham group and model group, **p<0.01; Comparisons between sham group and PHPS1 group, **p<0.05; Comparisons between model group and PHPS1 group, **p<0.01.

Results of oxidative stress related proteins in mouse cardiomyocytes

The results of western blot showed that the relative protein expressions of P22, P47 and gp91 in the model group and PHPS1 group were significantly increased compared with the sham group. Compared with the model group, the relative protein expressions of P22, P47 and gp91 in the PHPS1 group were significantly increased (Figure 5), which indicated that SHP2 could improve the production of oxidative stress in sepsis induced myocardial injury.

Figure 5. Western blot results of oxidative stress related proteins in myocardial cells of mice in each group. (A) Western blot results of P22, P47 and gp91 in mouse cardiomyocytes in each group; (B) Statistical plots of P22, P47 and gp91 expression levels in mice cardiomyocytes in each group, and the data was shown as X±S. Comparisons between sham group and model group, **p<0.01; Comparisons between sham group and PHPS1 group, **p<0.01; Comparisons between model group and PHPS1 group, **p<0.01.

Results of the apoptosis-related proteins and the apoptosis of cells

Western blotting results showed that the relative protein expressions of Bax, sheared caspase-3 and sheared caspase-12 in the model group and PHPS1 group were significantly increased compared with the sham group. Compared with the model group, the relative protein expressions of Bax, cleaved-caspase-3 and cleaved-caspase- 12 in the PHPS1 group were significantly increased. The results of TUNEL assay showed that the number of apoptotic cells in the model group and PHPS1 group was significantly higher than that in the sham group. Compared with the model group, the number of apoptotic cells in the PHPS1 group was significantly increased (Figure 6). The results manifested that SHP2 inhibited cardiomyocyte apoptosis in septic myocardial injury.

Figure 6. Results of the expression of apoptosis-related proteins in mouse cardiomyocytes and the cell apoptosis condition. (A) Western blot results of Bax, cleaved-Caspase-3 and cleaved-Caspase-12 in mouse cardiomyocytes in each group; (B) Statistical plot of Bax, cleaved-Caspase-3 and cleaved-Caspase-12 expression levels in mouse cardiomyocytes in each group; (C) Plot of TUNEL assay results; (D) Apoptotic cell count statistics, data was shown as X±S. Comparisons between sham group and model group, **p<0.01; Comparisons between sham group and PHPS1 group, **p<0.01; Comparisons between model group and PHPS1 group, **p<0.01.

In vitro experimental results

Western blot results showed that the expression of p-SHP2 protein in Group c was lower than that in Group b, but the expression level of p-Src, P22, P47, Bax and Caspase-3 protein in Group c was higher than that in Group b. The expression of p-SHP2 protein in Group d was higher than that in Group e, but the expression of p-Src protein was lower than that in Group c. The differences between the relative protein expressions of P22, P47, Bax, and cleaved-caspase-3 were all narrowed between groups d and e after the addition of the antioxidant NAC (Figure 7). These results suggested that SHP2 inhibited the apoptosis of cardiomyocytes induced by oxidative stress in septic myocardial injury by inhibiting Src/ERK pathway (Figure 8).

Figure 7. In vitro experiment results. (A) Western blot results plots of p-SHP2, p-Src, P22, P47, Bax, and cleaved-Caspase-3; (B) Statistical plots of p-SHP2, p-Src, P22, P47, Bax, and cleaved-Caspase-3 expression levels, the data was shown as X±S. Comparisons between LPS group and LPS+PHPS1 group, **p<0.01; Comparisons between LPS+NAC group and LPS+PHPS1+NAC group, **p<0.01, *p<0.05, nsp>0.05.

Figure 8. Schematic diagram of myocardial injury induced by SHP2 in sepsis.

Groups and treatments

Mice groups and mice model

Eighteen 6-weeks-old SPF grade male C57BL/6J mice (C57BL/6JNifdc, Beijing Vital River Laboratory Animal Technology Co., Ltd., China) were reared adaptively for one week, during which all the mice had free access to food and water. One week later, the mice were randomly divided into sham group, model group and PHPS1 group, and each group consisted of 6 mice. The model of myocardial injury induced by sepsis was established by intraperitoneal injection of LPS (20mg/kg) in the model group and the PHPS1 group. The mice in the sham group were given intraperitoneal injection of normal saline (20mg/kg). The mice in PHPS1 group were intraperitoneally injected with LPS and PHPS1(3 mg/kg) at the same time. The animal experiments in this study have been approved by the Medical Ethics Committee of Hebei General Hospital. Ethics Lot Number: 202376.

Cells treatments and groups

Five suckling C57BL/6 mice (J006), 1- 3 days old, were purchased from Nanjing Junke Bioengineering Co., Ltd. (China. After anesthesia, the suckling mice were disinfected with 75% ethanol. Under sterile conditions, the thoracic cavity of the mice was exposed with sterile ophthalmic scissors, and the hearts of the suckling mice were taken out and washed with PBS. The atrium and surrounding tissue vessels of suckling mice were stripped and washed again. The myocardial tissues were cut into tissue fragments of about 1mm3 with ophthalmic scissors, and were lysed with lysis solution containing trypsin and type II collagenase. After lysis, the supernatant was centrifuged and obtained. Natural precipitation, discard the supernatant, add about 5ml of digestive solution, digest at 37° C for 20min (shake a few times every 2min), blow with a pipette for 1min, suck out the incomplete heart fragments into another centrifuge tube, add 2ml of cold culture medium to terminate digestion, 1000 rpm for 5min, discard the supernatant, add 2ml of D-Hanks solution to the precipitate, 1500 rpm for 10min, discard the supernatant, add 2ml of culture medium to the precipitate, and make a cell suspension by pipette. Then, the cells were cultured in DMEM medium (3-2010, Jiangsu CHI Scientific Biological Technology Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin at 37° C with 5% CO2, and LPS, PHPS1((HY-112368, MedChemExpress, USA) and NAC (50303ES05, Yeasen Biotechnology (Shanghai, China) Co., Ltd.) were added into the medium respectively when the cell fusion reached 80%. Therefore, the cells were then divided into five groups: Group a (without LPS, PHPS1 or NAC), Group b (with LPS only), Group c (with LPS and PHPS1), Group d (with LPS and NAC) and Group e (with LPS, PHPS1 and NAC).

Methods

Echocardiogram detection

The mice in each group were anesthetized with 50 mg/kg sodium pentobarbital, and the mice were observed after being given sodium pentobarbital, and the mice were kept until the mice were breathing steadily, their voluntary activities were reduced, their muscle tone was weakened, and the muscles were relaxed. Cardiac ultrasound was examined with MX250 ultrasound probe under the Vevo3100 high-resolution small animal ultrasound imaging system, and the probe was placed in the short axis papillary muscle section of the left heart of the mouse to obtain M-mode echocardiography, the probe should not over compress the chest wall of the mouse, so as not to cause respiratory and cardiac arrest, and the ratio of the left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS) and E/A ratio were used to evaluate the changes in cardiac function in mice.

HE staining

After the mouse heart tissue was fixed in paraformaldehyde fixative solution for 48 hours, the sliced heart tissue was placed in an embedding box and placed in 4% paraformaldehyde fixative solution, and then dehydrated and transparent and sectioned. The thickness of the slice is 3-4 um. Place the slide rack containing tissue slides in a 60° C oven for 45 min, deparaffinize 2 times, 15 min each time, then pass through absolute ethanol for 10 seconds, 95% ethanol for 10 seconds, 80% ethanol for 10 seconds, and then wash the slides thoroughly with clean water for 1 time; Then the tissue was soaked in hematoxylin for 2 minutes, rinsed with tap water, differentiated with ethanol hydrochloride for 5 seconds, and returned to blue for 1 min; Then use eosin treatment for half a minute, rinse with tap water; Then dehydrate and use xylene transparently. The dehydrated transparent finished slides are placed in a fume hood to dry and neutral gum mounted, and the myocardial structure is observed under a microscope and photographed.

Transmission electron microscope

Hearts were taken from mice, washed with PBS, placed on an ice box, aspirated with a disposable dropper, and fixed with 2.5% glutaraldehyde solution. With 0.1 mol· L-1 phosphate buffer, transfer the samples to 4° C and fix with 1% osmium acid for 3 h. With 0.1 mol· L-1 phosphate buffer, samples were dehydrated with gradient ethanol: 30% ethanol for 15 min, 50% ethanol for 15 min, 70 ethanol uranyl acetate overnight, 80% ethanol for 15 min, 90% ethanol: 90% acetone (1:1) for 15 min (all steps were performed at 4° C), then treated with 100% acetone for 15 min × 3 h (room temperature), pure acetone + embedding solution (1:1) for 2 h (room temperature), pure embedding solution in a 37° C oven for 3 h. The samples were then embedded in the embedding solution in a 37° C oven for 12 h and in a pure embedding solution oven at 60° C for 48 h. Semi-thin sections with a thickness of 1 μm were stained with sky blue and viewed under a light microscope to determine the location of the retina and the structure of the 10 layers. Ultrathin sections with a thickness of 80 nm were fished out, placed on a copper mesh of the supporting film, stained with lead citrate for 15 min, washed 3 times with distilled water, dried naturally, and observed by transmission electron microscopy.

Western blot

The cells were lysed with RIPA lysate (P0013K, Jiangsu Beyotime Biotechnology Co., Ltd., China). SDS-PAGE was used to analyze the protein, and after electrophoresis, the protein was transferred to PVDF membrane. Place the PVDF membrane into a TBST container packed with 5% skim milk and wash the membrane on a shaker for 10 min, pour out the TBST after 10 min, then add new TBST, wash the membrane again on the shaker for 10 min, pour out the TBST after 10 min, add clean TBST, and wash the membrane 3 times again on the shaker, then p-SHP2 antibody(ab62322, Abcam), p-Src antibody (ab40660, Abcam), p-ERK1/2 antibody (ab229912, Abcam), P22 antibody (ab75941, Abcam), P47 antibody (ab308256, Abcam), gp91 antibody (ab310337, Abcam), Bax antibody (ab32503, Abcam), Caspase-3 antibody (ab32351, Abcam), Caspase-12 antibody (ab62484, Abcam) and DAPDH antibody (ab8245, Abcam) were diluted in 1:1000 with TBST solution. After the washing is completed, the PVDF membrane is transferred to the HRP secondary antibody and incubated on a shaker for 2 hours at room temperature and in the dark. After the 2-hour secondary antibody incubation, wash the membrane again for 10 minutes each time, and develop color after the three times. Soak the membrane for 20 seconds using ECL chromogenic solution and develop color using a chemiluminescence imaging system, and the WB band of the protein of interest can be seen. Protein expression was measured by the gray value of the protein band.

TUNEL staining

The heart tissue was deparaffinized with xylene, dehydrated with gradient ethanol, and 0.1% Triton X-100 was prepared with 0.1% sodium citrate, and then 50 μl was added dropwise to each section, and then rinsed with PBS 3 times for 5 min each time after 8 min at room temperature. Enzyme solution and label solution were prepared 1:9 on ice to make TUNEL reaction. Wipe the specimen around it clean and add 50 μl of TUNEL reaction dropwise. Incubate at 37° C with moisture and protection from light for 1 h, and then rinse the specimens with PBS for 3 times for 5 min each time. Add DAPI dropwise until the tissue is completely covered, and counterstain in the dark for 3 min. Rinse the specimen 3 times with PBS for 5 min each. The slices are removed and fluorescent quencher dropwise is added to seal the slices. Observe the staining effect under a microscope and take photos.

Statistical analysis

All experiments in this study were repeated three times, and the data were statistically analyzed using SPSS 23.0 software. The measurement data was represented by (X±S). The differences between two groups of data were tested by t-tests, and the differences among multiple groups were compared using one-way ANOVA. P<0.05 was considered statistically significant.