TRIM62 knockdown by inhibiting the TLR4/NF-κB pathway and NLRP3 inflammasome attenuates cognitive impairment induced by diabetes in mice

Abstract

The tripartite motif 62 is an E3 ubiquitin ligase protein that regulates cellular processes, including differentiation, immunity, development and apoptosis, leading to various disease states, such as cancer and inflammatory diseases. However, the role and mechanism of the tripartite motif 62 in the process of diabetic-induced cognitive impairment have not been reported. Therefore, the aim of this study was to investigate the role and mechanism of the tripartite motif 62 in diabetic-induced cognitive impairment. The results showed that the expression of the tripartite motif 62 was up-regulated in diabetic mice. Silencing of TRIM62 increased body weight and decreased fasting blood glucose in diabetic mice. In addition, knockdown of the tripartite motif 62 inhibited STZ-induced inflammation, apoptosis and oxidative stress. Further studies showed that the TLR4/NF-κB pathway and NLRP3 inflammasomes were involved in the regulation of diabetic mice by the tripartite motif 62. More importantly, inhibition of the tripartite motif 62 improved cognitive impairment and learning ability in mice. In conclusion, inhibition of TRIM62 inhibits STZ-induced inflammation, cell apoptosis and oxidative stress, and improves the cognitive ability of mice. Therefore, the tripartite motif 62 may be an important target for the treatment of diabetes-induced cognitive impairment.

Introduction

Diabetes mellitus (DM) is a metabolic disorder that can cause many complications, such as chronic kidney disease, retinopathy and peripheral neuropathy.⁽¹⁾ At present, cognitive impairment is considered as a new type of complication of diabetes, which has attracted more and more attention.⁽²⁾ The aetiology of cognitive impairment caused by diabetes has many factors, including brain insulin resistance, cerebral microvascular injury, neuroinflammation and brain region-specific metabolic disorders.^(3,4) The brain is the organ most susceptible to glucose fluctuations and inflammation. Insulin resistance and inflammation caused by diabetes can lead to neurotransmitter synthesis disorders and neuronal plasticity disorders, leading to cognitive impairment.⁽⁵⁾ In addition, changes in blood sugar significantly increase oxidative stress, which can also promote cognitive impairment.⁽⁶⁾ Therefore, inflammation and oxidative stress play an important role in diabetes-induced cognitive impairment.

The tripartite motif (TRIM) family is a group of highly conserved E3 ubiquitin ligase proteins. TRIM protein can cause many different disease states by regulating cellular processes, including cancer, viral infections, inflammatory diseases and muscle diseases.⁽⁷⁾ TRIM62, also known as DEAR1, is a member of the TRIM family and can catalyse autoubiquitination in vitro and in vivo.^(8,9) Like other TRIM proteins, TRIM62 is involved in the regulation of differentiation, immunity, development and apoptosis. As a new tumor suppressor, TRIM62 can inhibit the proliferation, migration and invasion of lung adenocarcinoma cells.⁽¹⁰⁾ In addition, studies have found that TRIM62 induces the expression of NF-κB and AP-1 through the TRIF branch of the TLR4 signaling pathway, thereby participating in the regulation of immunity.⁽¹¹⁾

Toll-like receptor 4 (TLR4) is a member of the pattern recognition receptor (PRR) family. TLR4 activated by exogenous or endogenous ligands mediates a variety of inflammatory responses and plays a key role in ischemia/reperfusion (I/R) injury, neurodegenerative diseases and neurological diseases.⁽¹²⁾ TLR4 is widely distributed in the brain, and TLR4 in the central nervous system is involved in memory, learning disabilities and cognitive decline.^(13,14) Studies have shown that inhibiting TLR4 activation reduces the neuroinflammation and cognitive impairment induced by lipopolysaccharide.⁽¹⁵⁾ NF-κB participates in the inflammatory response and immune response process, and is related to the pathogenesis of many inflammatory diseases.⁽¹⁶⁾ In type 2 diabetic rats, inhibiting NF-κB activation reduced the levels of IL-6 and TNF-α, and improved learning and memory.⁽¹⁷⁾ Therefore, we speculate that the TLR4 signaling pathway may play an important role in cognitive impairment.

This study aimed to explore the role and mechanism of TRIM62 in the pathogenesis of cognitive impairment caused by diabetes. Our results revealed the basic role of TRIM62 in cognitive impairment and provided a potential drug target for the clinical treatment of cognitive impairment caused by diabetes.

Materials and Methods

High-fat diet/STZ-induced diabetes model in mice

Thirty four-week-old male C57BL/6J mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Ten mice were randomly selected to be fed a normal diet (control) as the control group (n = 10), and the remaining mice were fed HFD (60 kcal% fat). Four weeks later, mice in the HFD group were injected with 1% STZ (40 mg/kg) intraperitoneally, three times within a week to induce diabetes in the mice. The fasting blood glucose (FBG) of the T2DM rats was confirmed with an Optium Xceed FBG meter (Abbott Pharmaceutical Co., Ltd., Lake Bluff, IL). Mice with blood glucose >11.1 mmol/L were marked as diabetic mice. Diabetic mice were divided into three groups, one group without any treatment (n = 10), one group was injected with a sh-TRIM62 lentivirus (n = 10) and the other group was treated with Z-AVD (a caspase inhibitor). Mice in the TRIM62-silenced STZ group was additionally injected with 2 μl TRIM62 shRNA lentivirus using a 27-gauge needle and a syringe, with a four min-period of 0.5 L/min speed, bilaterally into hippocampus. All mice were kept in an SPF animal laboratory with a humidity of 60–65% and a temperature of 21–26°C. At the end of the experiment, the mice were euthanised and the hippocampus tissue was dissected for subsequent experiments. All animal experiments were approved by the ethics committee of Shaanxi Provincial People’s Hospital, and were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Morris water maze test

Before the end of the experiment, the mice were tested for their learning and memory ability using the Morris water maze experiment. The maze is divided into four quadrants, one of which has a platform located 1 cm below the water surface. Before training, put all mice were placed in the pool to swim freely for 1 min to familiarized themselves with the environment. During the training period, the mice were put into different positions each time, and the escape latency and path of the mice looking for the platform were recorded. The training period was 4 days, and the space exploration experiment was carried out on the fifth day. The platform was then removed and record the percentage of time the mouse spent in the quadrant to exploring the target platform, the number of times the mouse passed through the position of the front platform, and the length of the path used were recorded. All data were recorded by a computer tracking system.

Enzyme-linked immunosorbent assay

The contents of glycosylated haemoglobin (HbA1c), IL-6, and TNF-α in mouse serum were determined by ELISA kit. The content detection of GSH-PX, GSH, SOD, and MDA was completed according to the manufacturer’s instructions of the ELISA kit, and the content of each factor in the hippocampus tissue of each group of mice was determined.

Western blot analysis

The tissues were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors, and the supernatant was collected. Then, SDS loading buffer was added and samples were heated for 10 min for protein denaturation. The protein samples were loaded on a 10% SDS-PAGE gel for electrophoresis, and the protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk powder for 1 h, incubated with the primary antibody overnight at 4°C, incubated with the secondary antibody at room temperature for 2 h, and then washed with TBST. Enhanced chemiluminescence (ECL) reagents were used to observe the immune complexes on the membrane, and the ImageJ analysis system was used to quantify the luminosity of the bands.

Statistical methods

The results of three independent replicate experiments were expressed as mean ± SD. Use SPSS 19.0 software one-way analysis of variance and t test to compare the data statistically and to assess the differences between the groups. P<0.05 was considered statistically significant.

Results

Silencing of TRIM62 increases body weight and decreases fasting blood glucose in diabetic mice

Compared with the control group, the body weight of the STZ group mice was decreased significantly. It should be noted that at the end of siTRIM62 treatment, the body weight was increased significantly compared with STZ group (Fig. 1A). After 20 days of STZ treatment, the blood glucose of the model group was significantly higher than that of the control group, and the blood glucose level decreased significantly after siTRIM62 treatment (Fig. 1B). The blood HbA1c of STZ group mice was also significantly increased, and the blood HbA1c level of STZ group mice was significantly decreased with treatment siTRIM62 (Fig. 1C). Compared with the control group, STZ significantly promoted the expression of TRIM62 (Fig. 1D). These data suggest that siTRIM62 treatment has beneficial glucose metabolism regulation effects on increasing body weight, blood glucose and blood HbA1c levels in the STZ-induced diabetic mouse model.

Fig. 1.

Silencing of TRIM62 increases body weight and decreases fasting blood glucose in diabetic mice. Mice in the control group (n = 10) were intraperitoneally injected with normal saline. Streptozotocin (STZ) saline was injected intraperitoneally in type 2 diabetic mice. Two weeks after the onset of diabetes, mice were divided into 2 groups: T2DM mice (n = 10), and lentivirus T2DM mice injected with sh-TRIM62 (n = 10). (A) Body weight, (B) blood glucose, (C) glycosylated hemoglobin (HbA1c) in control, STZ and STZ + sh-TRIM62-treated DM mice. (D) The expression of TRIM62 in control, STZ and STZ + sh-TRIM62-treated DM mice. * means compared with control group p<0.05, ** means compared with control group p<0.01, and # means compared with STZ group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Silencing of TRIM62 inhibits diabetic induced cognitive impairment

In the Morris water maze assay, the escape latency of the STZ group mice significantly increased after 2–4 days of training compared with the control group. Compared with the STZ group, siTRIM62 treatment significantly shortened the escape latency (Fig. 2A). In addition, on the fifth day, the time in the target quadrant in diabetic mice decreased significantly compared with control mice (Fig. 2B). Compared with the control group, the frequency of animals crossing the previous platform location was significantly reduced in diabetic mice (Fig. 2C). These changes were significantly reversed by siTRIM62 treatment in diabetic mice, and the results were not significantly different from those of control mice (Fig. 2B and C). In addition, compared with the control group, the mean path length of the STZ group mice increased significantly, while siTRIM62 treatment reversed this effect, and the mean path length was significantly shorter than that in STZ group mice (Fig. 2D). In conclusion, these results suggest that siTRIM62 may reduce cognitive impairment in diabetic mice.

Fig. 2.

Silencing of TRIM62 inhibits diabetic induced cognitive impairment. Mice in the control group (n = 10) were intraperitoneally injected with normal saline. Streptozotocin (STZ) saline was injected intraperitoneally in type 2 diabetic mice. Two weeks after the onset of diabetes, mice were divided into 2 groups: T2DM mice (n = 10), and lentivirus T2DM mice injected with sh-TRIM62 (n = 10). (A) Escape latency, (B) percentage of time spent in the target quadrant, (C) the number of times animals crossed the former platform location for day 5 and (D) mean path length, in control, STZ and STZ + sh-TRIM62-treated DM mice. ** means compared with control group p<0.01, and ^# means compared with STZ group p<0.05.

Silencing of TRIM62 inhibits diabetes-induced inflammation and apoptosis

Next, we explored the role of TRIM62 on inflammation and apoptosis. The results show that STZ significantly promoted the levels of IL-6, IL-8, and TNF-α in mice, while inhibition of TRIM62 significantly reduced the increase of STZ-induced inflammatory factors (Fig. 3A). Further study revealed that siTRIM62 inhibited the expression of iNOS and COX-2 compared with the STZ group (Fig. 3B). These results indicated that silencing of TRIM62 inhibits diabetes-induced inflammation.

Fig. 3.

Silencing of TRIM62 inhibits diabetes induced inflammation and apoptosis. Hippocampal regions of three groups of mice were taken respectively. (A) The levels of IL-6, IL-8, and TNF-α, (B) The protein expression of iNOS and COX-2, (C–E) The protein expression of caspase-3, Bax, and Bcl-2 in control, STZ, STZ + sh-TRIM62-treated and Z-VAD DM mice. ** means compared with control group p<0.01, and # means compared with STZ group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Our research also explored the role of TRIM62 on apoptosis in mice. We used caspase inhibitor (Z-VAD) to evaluate the effect of TRIM62 on cell apoptosis. As presented in Fig. 3C, STZ-induced the apoptosis, as well as increased the expression of Bax and caspase-3, and decreased the expression of Bcl-2. On the contrary, TRIM62 significantly reversed these effects, consistent with the effect of Z-VAD. (Fig. 3C–E). These results indicated that siTRIM62 inhibited the apoptosis in diabetic mice.

Silencing of TRIM62 reduces oxidative stress induced by diabetes mellitus

The results show that STZ significantly inhibited the expression of GSH-Px and SOD, while inhibition of TRIM62 significantly increased the STZ-induced reduction (Fig. 4A–C). The ELISA results showed that the levels of GSH-Px (Fig. 4D), SOD (Fig. 4E) and GSH (Fig. 4F) and were significantly lower in STZ mice than those in the control group. However, siTRIM62 treatment significantly increased the levels of GSH-Px and SOD compared with STZ group mice (Fig. 4D–F). In addition, compared with the control group, the MDA level of STZ group mice was significantly increased, and siTRIM62 treatment significantly reduced the MDA content of STZ group mice (Fig. 4G). These results suggest that siTRIM62 may reduce the oxidative stress level in diabetic mice.

Fig. 4.

Silencing of TRIM62 reduces oxidative stress induced by diabetes mellitus. Hippocampal regions of three groups of mice were taken respectively. (A–C) Protein expression levels of GSH-PX and SOD were detected by Western blot analysis. ELISA was performed to determine the levels of (D) GSH-PX, (E) SOD, (F) GSH, and (G) MDA in control, STZ and STZ + sh-TRIM62-treated DM mice. ** means compared with control group p<0.01, and ^# means compared with STZ group p<0.05, ^## means compared with STZ group p<0.01. DM, diabetes mellitus; GSH, glutathione; GSH-PX, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde.

The effect of TRIM62 on changes of hippocampal tissue in diabetic mice

Western blot was used to measure the protein expression of eNOS, PPARγ, and AMPK (Fig. 5A). Compared with the control group, the protein expression of eNOS, PPARγ, and AMPK of diabetic mice was significantly decreased (Fig. 5B–D), while siTRIM62 treatment significantly increased he protein expression of eNOS, PPARγ, and AMPK in diabetic mice (Fig. 5B–D). These above results show that TRIM62 plays an important role in the changes to hippocampal tissue in diabetic mice.

Fig. 5.

The effect of TRIM62 on changes of hippocampal tissue in diabetic mice. Hippocampal regions of three groups of mice were taken respectively. (A) Protein expression levels of eNOS, PPARγ, and AMPK were detected by Western blot analysis. Densitometric analysis of Western blotting results was performed to quantify the protein levels of (B) eNOS, (C) PPARγ, and (D) AMPK in control, STZ and STZ + sh-TRIM62-treated DM mice. * means compared with control group p<0.05, and ^# means compared with STZ group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Silencing of TRIM62 inhibits activation of the TLR4/NF-κB pathway and NLRP3 inflammasomes

In order to verify the mechanism of TRIM62 in diabetes-induced cognitive impairment of mice, we speculated that the TLR4/NF-κB pathway and NLRP3 may be involved in this process. We found that the protein expression of TLR4, p-p65, and NLRP3 of diabetic mice was significantly increased compared with the control group (Fig. 6). Interestingly, siTRIM62 significantly decreased the protein expression of TLR4, p-p65, and NLRP3in diabetic mice (Fig. 6). In conclusion, inhibition of TRIM62 may improve the cognitive function and learning ability of mice by regulating the TLR4/NF-κB pathway and NLRP3 inflammasomes.

Fig. 6.

Silencing of TRIM62 inhibits activation of TLR4/NF-κB pathway and NLRP3 inflammasomes. Hippocampal regions of three groups of mice were taken respectively. (A) Protein expression levels of TLR4, p-p65, p65, and NLRP3 were detected by Western blot analysis. Densitometric analysis of Western blotting results was performed to quantify the protein levels of (B) TLR4, (C) p-p65/p65, and (D) NLRP3 in control, STZ and STZ + sh-TRIM62-treated DM mice. ** means compared with control group p<0.01, and ^# means compared with STZ group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Discussion

Cognitive impairment is one of the most serious complications of diabetes, and involves changes in brain structure and pathology.⁽¹⁸⁾ There is increasing evidence that neuroinflammation and oxidative stress play a key role in cognitive impairment. Especially the inflammation of the hippocampus, because it is the centre of learning and memory processing in the brain, damage to the hippocampus may cause damage to various cognitive fields.⁽¹⁹⁾ Inflammation of the central nervous system is usually a protective response. However, chronic inflammation over a long period of time can be harmful. It may cause the degeneration and premature death of specific neuron groups, and promote the progression of cognitive impairment.^(5,20) Reducing neuroinflammation may be an effective strategy in the progression of cognitive impairment. Our study found that silencing TRIM62 inhibited diabetes-induced inflammation and apoptosis, which improved the cognitive impairment in mice.

As we all know, the distinguishing feature of diabetes is high blood sugar. Hyperglycemia can increase the mitochondrial respiration of endothelial cells and astrocytes, which promote the production of ROS and oxidative stress. Elevated ROS upregulates the expression of inflammatory cytokines, and continuous inflammation can cause cognitive impairment.⁽²¹⁾ Neuroinflammation and oxidative stress are key processes involved in the pathophysiology of countless brain diseases, and are closely related to the development of cognitive impairment. Aged mice show increased sensitivity to cognitive impairment after the occurrence of systemic inflammation related to persistent oxidative stress and neuroinflammation.⁽²²⁾ Improving lipopolysaccharide-induced neuroinflammation and oxidative stress has been shown to alleviate memory impairment in mice.⁽²³⁾ As a kind of ubiquitin ligase, TRIM62 has a certain role in inflammation. TRIM62 promotes inflammation-induced muscle atrophy by activating the AP-1 signal transduction pathway, which may be a new factor leading to sustained inflammation.⁽²⁴⁾ Silencing TRIM62 has a protective effect in ischemic stroke by reducing the neuroinflammation regulated by NLRP3.⁽²⁵⁾ This is very similar to the results of our study, as silencing TRIM62 alleviated diabetes-induced neuroinflammation and oxidative stress.

In the central nervous system, microglia are the phagocytes in the brain. These cells play an important role in injury responses, pathogen defence and neural circuit development and shaping.⁽²⁶⁾ TLR4 is expressed in neurons and microglia, and is highly involved in neuroinflammation during central nervous system injury.⁽²⁷⁾ Studies have shown that inhibition of TLR4 can induce the polarisation of M2 microglia to provide neuroprotection in Alzheimer’s disease.⁽²⁸⁾ In addition, TLR4 mediated diabetes-induced cognitive impairment in mice.⁽²⁹⁾ Therefore, TLR4 may be an important molecule in the treatment of cognitive disorders. In neuroinflammation, the activation and transduction of TLR4 pro-inflammatory signals mainly involves the TLR4/caspase-8/caspase-3/NF-κB signalling pathway and the TLR4/MyD88/TRAF6/NF-κB signalling pathway.⁽³⁰⁾ Activated NF-κB can induce cytotoxic products, aggravate inflammation and oxidative stress, as well as promote cell apoptosis, leading to cell dysfunction or death.⁽³¹⁾ In addition, the NLRP3 inflammasomes have been extensively studied in neuroinflammation and neurodegenerative disease.⁽³²⁾ Inhibition of NLRP3 can prevent the adverse effects of immune stimulation on synaptic plasticity and neuronal morphology.⁽³³⁾ In autoimmune encephalomyelitis (EAE) mice, inhibition of NLRP3 has been shown to ameliorated cognitive deficits.⁽³⁴⁾ Therefore, we measured the expression levels of TLR4 and NLRP3 in this study. The results showed that TRIM62 silencing inhibited the activation of the TLR4/NF-κB pathway and the NLRP3 inflammasome. However, the mechanism of silencing TRIM62 to activate the NLRP3 inflammasome still needs to be further explored.

In conclusion, we have demonstrated for the first time the potential role of TRIM62 in diabetes-induced cognitive impairment in mice. Silencing TRIM62 reduced neuroinflammation in mice by inhibiting the activation of the TLR4/NF-κB pathway and NLRP3 inflammasome, and moreover inhibited diabetes-induced cognitive impairment. TRIM62 may be a potential target in the treatment of cognitive impairment.

Funding

The study was funded by Key Research and Development Program of Shaanxi (2021SF-073).

Availability of Data and Materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

No potential conflicts of interest were disclosed.