Yun-Feng Wei , Dao-Ning Guo , Qiong Liu , Yan Dai , Qian Ding ?

1. Department of Ultrasound Medicine, Mianyang Central Hospital in Sichuan Province, Mianyang, Sichuan Province, 621000 2. Department of Ophthalmology, Mianyang Central Hospital in Sichuan Province, Mianyang, Sichuan Province, 621000

Keywords:Diabetic retinopathy High mobility group box 1 Infalmmatory response Apoptosis

ABSTRACT Objective: To study the effect of inhibiting high mobility group box 1 (HMGB1) gene expression on ultrasonic blood flow characteristics, inflammatory response and apoptosis in diabetic retinopathy rats. Methods: Male SD rats were chosen and randomly divided into control group, model group and HMGB1 inhibition group, the latter two groups were given intraperitoneal injection of streptozotocin to establish the diabetes model, and HMGB1 group were given tail vein injection of HMGB1-shRNA lentivirus for intervention. The peak endsystolic velocity (PSV), peak end-diastolic velocity (EDV), resistance index (RI) and pulsatility index (PI) of central retinal artery as well as the HMGB1, bcl-2, bax and cleaved caspase-3 expression and TNF-α, MCP-1 and ICAM-1 contents in retinal tissues were measured at 4 weeks after intervention. Results: The PSV and EDV levels as well as bcl-2 expression in the model group were lower than those in the control group, while the RI and PI levels as well as HMGB1, bax and cleaved caspase-3 expression and TNF-α, MCP-1 and ICAM-1 contents were higher than those in the control group; the PSV and EDV levels as well as bcl-2 expression in the HMGB1 inhibition group were higher than those in the model group, while the RI and PI levels as well as HMGB1, bax and cleaved caspase-3 expression and TNF-α, MCP-1 and ICAM-1 contents were lower than those in the model group. Conclusion: Inhibition of HMGB1 gene expression can improve the blood flow of central retinal artery and inhibit the inflammatory response and apoptosis in diabetic retinopathy rats.?Corresponding author: DING Qian.

1. Introduction

Diabetic retinopathy (DR) is one of the common microvascular complications of diabetes and also one of the four clinical causes of blindness. The occurrence of the disease involves multiple factors and multiple steps, but the specific pathogenesis is still unclear. The pathological features of the local tissues of DR in the early stage include thickening of the basement membrane of blood vessels, lumen stenosis and damage of vascular endothelial cells, and there may be gradual abnormal angiogenesis and damage of ganglion cells in the later stage [1-2]. At present, factors related to the pathogenesis of DR are known to include hyperglycemia, blood glucose fluctuation, activation of inflammatory response, and apoptosis [3-4]. High mobility group box 1 (HMGB1) is an wadvanced inflammatory factor, which not only participates in the regulation of inflammatory response, but also stimulates apoptosis and angiogenesis. It has been reported in cell experiments that high glucose stimulation can increase the expression of HMGB1 in retinal vascular endothelial cells, while inhibiting the expression of HMGB1 can improve the apoptosis caused by high glucose [5]. Based on this, it is hypothesized that inhibition of HMGB1 can play a positive role in the treatment of DR. In order to verify this hypothesis, diabetic rats were taken as the objects in this experiment to specifically analyze the effect of inhibiting HMGB1 gene expression on ultrasonic blood flow characteristics, inflammatory response and cell apoptosis.

2. Materials and methods

2.1 Animals

Male SD rats, 8-10 weeks old, with a body mass of 220-250g, were purchased from Shanghai Maosheng Derivatives Technology Co., LTD., production license was SCXK (Shanghai) 2017-0004, the environment temperature was 22-24 ℃, humidity was 40-60%, food and water were freely consumed, and the light cycle was 12h daylight /12h darkness.

2.2 Adenovirus

HMGB1-shRNA lentivirus was from Shanghai Genechem Company.

2.3 Reagents and instruments

Streptozotocin (STZ) was from Sigma, and the monoclonal primary antibody of HMGB1, bcl-2, bax and cleaved caspase-3 were from Abcam. Elisa kits for TNF-α, MCP-1 and ICAM-1 were provided by Shanghai Elisa Company, VINNO6 LAB small animal ultrasound device was provided by Beijing Yeeran Company, and GelDox XR+ gel imager was provided by Bio-rad Company.

2.4 Animal grouping and intervention

SD rats were randomly divided into control group, model group and HMGB1 inhibition group, with 10 rats in each group. The model group and the HMGB1 group were modeled by intraperitoneal injection of STZ, which was as follows: STZ solution was prepared with 0.01mol/L citrate-sodium citrate buffer at pH4.5, followed by a single intraperitoneal injection of 55mg/kg STZ after overnight fasting for solids but not for liquids, and glucose was measured through the caudal vein 72 hours later, and the glucose ＞ 16.7mmol/L indicated successful modeling. Control group were given intraperitoneal injection of same volume of citrate - sodium citrate buffer. HMGB1 inhibition group were intervened on day 2 after successful modeling, and received HMGB1-shRNA lentivirus 1×109IU injection via the caudal vein, while the other two groups received 1mL saline injection via the caudal vein.

2.5 Ultrasonic evaluation of blood flow in the central retinal artery

After 4 weeks of intervention, 10% chloral hydrate 0.3mL/kg was injected intraperitoneally for anesthesia, and the flow parameters of the central retinal artery were measured by small animal ultrasound device, including peak end-systolic velocity (PSV), peak enddiastolic velocity (EDV), resistance index (RI) and pulsatility index (PI).

2.6 Western blot analysis of protein expression

After ultrasonic testing, the rats were executed, retina tissue was anatomized and added into RIPA lysate to extract the protein sample in it, the protein content was determined, then the sample containing 30μg protein was taken and added into the polyacrylamide gel for electrophoresis and electric transfer into NC membrane, the NC membrane was closed in the 5% skim milk for 1 hour at room temperature, and then the NC membrane was put in the monoclonal primary antibody of HMGB1, bcl-2, bax and cleaved caspase-3 for the night; on the second day, the NC film was washed, then put into HRP secondary antibody for 1 hour of incubation and finally exposed in the gel imager to get protein bands, and the expression levels were calculated according to the gray value of the bands.

2.7 Elisa detection of cytokine contents

The protein samples obtained in the previous step were taken, the contents of TNF-α, MCP-1 and ICAM-1 were detected by Elisa, and the contents of TNF-α, MCP-1 and ICAM-1 per mg of protein were calculated based on the protein contents.

2.8 Statistical methods

SPSS20.0 software was used to input data, and ANOVA was used for comparison of measurement data among the three groups. P＜0.05 indicated that the difference was statistically significant.

3. Results

3.1 Comparison of HMGB1 expression in retinal tissue among the three groups

Compared with that of the control group, HMGB1 expression in retinal tissue of the model group was significantly higher, and the difference was statistically significant (P＜0.05); compared with that of the model group, HMGB1 expression in retinal tissue of the HMGB1 inhibition group was significantly lower, and the difference was statistically significant (P＜0.05), as shown in Fig 1 and Table1.

Fig. 1 Protein bands of HMGB1 in retinal tissue of the three groups

Table1 Comparison of HMGB1 expression in retinal tissue among the three groups (n=10)

3.2 Comparison of ultrasonic blood flow parameters in central retinal artery among the three groups

Compared with those of the control group, the PSV and EDV levels in central retinal artery of the model group were significantly lower, while the RI and PI levels were significantly higher, and the differences were statistically significant (P＜0.05); compared with those of the model group, the PSV and EDV levels in central retinal artery of the HMGB1 inhibition group were significantly higher, while the RI and PI levels were significantly lower, and the differences were statistically significant (P＜0.05), as shown in Table 2.

3.3 Comparison of inflammatory factor contents in retinal tissue among the three groups

Compared with those of the control group, TNF-α, MCP-1and ICAM-1 contents in retinal tissue of the model group were significantly higher, and the differences were statistically significant (P＜0.05); compared with those of the model group, TNF-α, MCP-1 and ICAM-1 contents in retinal tissue of the HMGB1 inhibition group were significantly lower, and the differences were statistically significant (P＜0.05), as shown in Table 3.

Table 2 Comparison of PSV, PDV, PI and RI in central retinal artery among the three groups (n=10)

3.4 Comparison of apoptosis gene expression in retinal tissue among the three groups

Compared with those of the control group, bcl-2 expression in retinal tissue of the model group was significantly lower, while bax and cleaved caspase-3 expression were significantly higher, and the differences were statistically significant (P＜0.05); compared with those of the model group, bcl-2 expression in retinal tissue of the HMGB1 inhibition group was significantly higher, while bax and cleaved caspase-3 expression were significantly lower, and the differences were statistically significant (P＜0.05), as shown in Fig 2 and Table 4.

Fig. 2 Protein bands of bcl-2, bax and cleaved caspase-3 in retinal tissue of the three groups

4. Discussion

The activation of inflammatory response is the key pathological link that causes diabetic retinopathy. The large release of inflammatory cytokines during the activation of inflammatory response can on the one hand, cause endothelial cell injury, cause microcirculation disorder, and then cause local retinal hypoxia, and on the other hand, stimulate angiogenesis and cell apoptosis, and thus cause local abnormal retinal neovascularization and ganglion cell damage [6-7]. HMGB1 is an advanced inflammatory factor involved in the regulation of inflammatory response. Clinical studies have reported that the serum content of HMGB1 has significantly increased in both diabetic nephropathy and DR patients [8-9]; other basic studies reported that both the expression of HMGB1 in retinal tissues of diabetic rats and the expression of HMGB1 in retinal endothelial cells under high glucose stimulation increased [10-11].

In this experiment, it was observed that the expression of HMGB1 in retinal tissues increased after the establishment of diabetes model by intraperitoneal injection of STZ, which was consistent with previous reports by other scholars [10-11], and indicated that the high expression of HMGB1 was related to the occurrence of DR, and further suggested that HMBG1 might be an effective target for the treatment of DR. In GUO Yujing's cell experiments, inhibition of HMGB1 expression can significantly improve retinal endothelial cell apoptosis caused by high glucose, but it remains unclear whether inhibition of HBMG1 expression can play a role in retinal protection in diabetic rats. To this end, HMGB1-shRNA lentivirus was designed in the experiment, and lentivirus injection was adopted to suppressthe expression of HMBG1, lentivirus was injected via caudal vein after diabetes model was established, and it was observed that HMGB1 expression significantly reduced in retinal tissue after 4 weeks, meaning that HMGB1-shRNA lentivirus can significantly inhibit the expression of HMGB1 in the retinal tissue of diabetic rats.DR is a type of diabetic microvascular disease, and stenosis of local supplying vessels to the retina may occur during the development of the disease. The central retinal artery is the main supplying artery of the retina. During the course of DR disease, stenosis or even occlusion occurred in this artery, resulting in decreased blood flow in the artery and insufficient blood supply to the retina [12-14]. In this study, the blood flow characteristics of the central retinal artery were evaluated by ultrasonic device. In the model group, PSV and EDV of the central retinal artery were significantly decreased, while PI and RI were significantly increased, indicating that diabetic rats had decreased blood flow and increased resistance in the central retinal artery. After HMGB1-shRNA lentivirus injection via the caudal vein, PSV and EDV in the central retinal artery of the rats increased significantly, while PI and RI decreased significantly, indicating that inhibiting the expression of HMGB1 could improve the blood flow state of the central retinal artery of the diabetic rats, increase blood flow and decrease blood flow resistance.

Table 3 Comparison of TNF-α, MCP-1 and ICAM-1 contents in retinal tissue among the three groups (n=10)

Table 4 Comparison of bcl-2, bax and cleaved caspase-3 in retinal tissue among the three groups (n=10)

HMGB1 is an advanced inflammatory cytokine whose main biological activities include activation of inflammatory response and apoptosis. TNF-α, MCP-1 and ICAM-1 are known inflammatory cytokines associated with the occurrence of DR. TNF-α has proinflammatory activity, MCP-1 has chemotactic activity, and ICAM-1 has adhesive activity. In the retinal tissues of DR, the three are involved in the activation of inflammatory response by promoting the activation, chemotaxis and adhesion of inflammatory cells [15]; bcl-2 and bax are molecules involved in apoptosis regulation. In retinal tissue of DR, bcl-2 can inhibit apoptosis while bax can promote apoptosis, and both ultimately participate in apoptosis through the generation of downstream cleaved caspase-3 [16]. The analysis of the above inflammatory factors and apoptosis genes in retinal tissues of diabetic rats in this study confirmed that the contents of TNF-α, MCP-1 and ICAM-1, as well as the expression of bax and cleaved caspase-3 in retinal tissues of the model group increased, while the expression of bcl-2 decreased, indicating that there was excessive activation of inflammatory response and apoptosis in retinal tissues of diabetic rats. After HMGB1-shRNA lentivirus injection via the caudal vein, the contents of TNF-α, MCP-1 and ICAM-1 as well as the expressions of bax and cleaved caspase-3 in the retinal tissues of rats decreased, while the expression of bcl-2 increased, indicating that inhibiting the expression of HMGB1 had an inhibitory effect on the inflammatory response and cell apoptosis in the retina of diabetic rats.

In conclusion, inhibition of HMGB1 gene expression can improve the blood flow in the central retinal artery of DR rats and inhibit the inflammatory response and cell apoptosis. In the future, inhibition of HMGB1 is expected to be a target for the treatment of DR, but specific means for targeted inhibition of HMGB1 still need to be further studied.