Beneficial Effects of Echinacoside on Cognitive Impairment and Diabetes in Type 2 Diabetic db/db Mice

Keywords

echinacoside - diabetes - cognitive impairment - insulin resistance

Introduction

As a global public health issue, diabetes has adversely affected more than 500 million people with multiple chronic complications worldwide [1]. Particularly, cognitive dysfunction, intricately correlated with type 2 diabetes (T2D), is one of the most serious complications, affecting more than 90% of patients with diabetes [2]. Approximately 50% of patients with T2D experience cognitive decline, including structural and functional brain impairment, gray matter atrophy, faster brain aging, and worse control of memory and information-processing [3] [4] [5].

T2D-related cognitive diseases include asymptomatic cognitive decline, mild cognitive impairment, and dementia, with vascular dementia and Alzheimer’s disease (AD) being the most common types [6] [7]. Studies suggest a nearly 1.5 times higher incidence of AD in cases with preexisting diabetes than in others [8] [9]. Considering the correlation between AD and diabetes, researchers even indicated that AD could be referred to as “brain diabetes” or “type 3 diabetes” [10] [11]. Although the diabetes-specific drugs cannot slow or decrease cognitive dysfunction, previous studies have shown that metformin might reduce cognitive decline [7] [12]. Overall, with the prevalence and harmfulness of diabetic encephalopathy, more effective cures for diabetes-related cognitive dysfunction need to be continuously explored [13].

According to clinical trials and in vitro and in vivo experiments, natural products from plants are promising for the prevention and management of T2D-related complications [14] [15] [16] [17]. Cistanche tubulosa, the most commonly used tonic Chinese medicine, might alleviate AD and cerebral ischemic injuries [18] [19] [20]. Moreover, consistent with our previous finding [21], researchers have shown the beneficial effects of C. tubulosa on diabetes and diabetic complications in mouse models [22] [23] [24].

Identification of potentially effective components from natural herbs might provide sources for the development of new drugs for diabetic encephalopathy [25] [26] [27]. Echinacoside (ECH) is the most active component of C. tubulosa. Previous studies have shown ECH might be potentially promising for the treatment of depressive disorders, vascular dementia, cerebral ischemia, Parkinson’s disease, and AD [28] [29] [30] [31] [32]. ECH can freely cross the blood-brain barrier, exhibiting neuroprotective, anti-oxidative, anti-neuroinflammatory properties, and regulating apoptosis and autophagy [33]. However, effects of ECH on T2D-related cognitive dysfunction are still limited.

Hyperphosphorylation tau(p-tau) aggregation in neurofibrillary tangles (NFTs) is closely associated with cognitive decline in neurodegenerative disease [34] [35]. Several studies pointed out a close link between p-tau and glycogen synthase kinase-3 beta (GSK3β) [36] [37]. The phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT)/GSK3β is a classical pathway activated by insulin. Under physiological conditions, active AKT inhibits GSK3β to modulate the phosphorylation balance of tau [36]. Thus, the occurrence of p-tau is associated with insulin resistance [38] [39]. Bioinformatics analysis shows that CD44, a biomarker of astrocyte cells, is positively correlated with T2D and AD through mechanisms involving inflammation and insulin resistance [40]. Soluble CD44 secreted by glioblastoma cells induces neuronal degeneration through the activation of tau pathology in the brain [41]. The level of CD44 expression in the brain of db/db mice and its relationship with insulin resistance has not been well-reported. The current study aims to explore the potential effect of ECH in improving cognitive impairment in db/db mice, and on insulin resistance, p-tau, and CD44.

Herein, in this study, we focused on the potentially beneficial regulation of ECH on cognitive impairment in db/db mice, one representative mice model of type-2 diabetes. Besides, for a better understanding of the behind mechanisms, we evaluated the effects of ECH on insulin resistance, hyperphosphorylation tau(p-tau) aggregation, and CD44, three important markers or events highly correlated to T2D and neurodegeneration disease [38] [39] [40].

Methods

Experimental animals

The Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University granted approval for the experimental procedures (Issue number: 20190517). Our animal care and handling practices strictly adhered to the declaration of Helsinki and the guidelines set forth by Renmin Hospital, Wuhan University. Male mice (10-week-old, C57BLKS/J db/db) and specific pathogen-free db/m mice were obtained from Nanjing University, Nanjing Institute of Biomedicine, China.

Main instruments and reagents

Aqueous solution of (2 mg/mL; ECH No.190906, China) was obtained from Shanghai Medical Science Company. Afterwards, the solution was stored at 4°C in a dark environment. Insulin ELISA kit (Abcam ab, 277390), Hematoxylin and Eosin Staining Kit (Beyotime, C0105S), Nissle Staining Kit (Solarbio, G1436), BCA protein concentration detection kit, sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparation kit (Epizyme, PG112), TRIzol reagent (Thermofisher, 15596026), One-step gDNA Remover (Servicebio, G3337), SYBR Green Supermix (Servicebio, G3326), and BeyoECL Plus (Beyotime, P0018M) were obtained.

Animal grouping and treatment

To conduct the experiments, a group of mice (10 weeks old) was kept in isolation for 1 week and provided with adaptive feeding for an additional week. Then, the mice were divided randomly into three groups: the control group (db/m, n=7), the diabetic model group (db/db, n=7), and the ECH-treated group (db/db+ECH, n=7). At 12 weeks, the normal control group and mice in the diabetic model group were both given normal saline (0.05 mL/10 g) intragastrically. However, the mice in the ECH group received a daily dose of 300 mg/kg of ECH, administered intragastrically [21]. Throughout the 14-week experimental period, the mice had unrestricted access to food and water. After 14 weeks of intervention, the mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (100 mg/kg) to collect blood samples. The serum was then separated and immediately stored at −80°C for further analysis after inserting a capillary needle. To eliminate blood residue, the brain was perfused with phosphate buffered saline (PBS), and any excess PBS was removed using filter paper. The hippocampus regions of the brains were obtained and subjected to histological examination, western blotting, and RT-PCR methods.

General conditions

Every 2 weeks, the mice were carefully weighed and their blood glucose levels were accurately measured. Upon reaching the end of week 26, following an 8-h fasting period, blood samples were collected from the mice through punctures made on their tail veins. The fasting plasma glucose (FPG) levels were then measured using a blood glucose meter (Johnson & Johnson, New Brunswick, NJ, USA). While performing the oral glucose tolerance test (OGTT) experiment, mice were administered glucose (2 g/kg) by gavage. The blood glucose values of mice were measured at 0 min before glucose administration, 15, 30, 60, and 120 min after glucose administration, and the area under the curve (AUC) of time blood glucose value was calculated. Fasting insulin levels (FINS) were measured using the ELISA kit (Abcam ab, 277390) according to the manufacturer's instructions. A standard curve was constructed using the concentration and optical density values of the standard sample, enabling the calculation of the sample concentration. The insulin resistance index (HOMA-IR) was calculated using the following formula: HOMA-IR=FPG×FINS/22.5 (Since the IR formula values are non-normally distributed, their natural logarithms are taken for statistical treatment).

Morris Water Maze test

Morris's water maze test was used to test spatial memory and learning ability after 14 weeks of continuous intervention. The water maze device consisted of a circular swimming pool and a platform. A mixture of water and milk powder (23±1 ℃) was added to the pool to make it opaque. The pool was divided into four quadrants, east, south, west, and north, and one of the quadrants was used as the target quadrant to place a 6 cm diameter circular platform that was 1 cm under the water surface. In the 5-day hidden platform experiment, the mice were allowed to enter the water with their heads facing the wall of the pool, and then to explore the water freely for 60 s. The time it took the mice to find the platform from the time they entered the water (escape latency) was recorded. If the mice did not find the platform within the 60 s, they were led to the platform and allowed to stay there for 15 s. Training trials were performed four sessions a day at intervals of more than 30 minutes, and entering the water from four different quadrants. On day 6, the underwater platform was removed, and each mouse was placed in the quadrant opposite to the target quadrant (where the underwater platform was located). The target platform crossings, time spent in the target quadrant, and swimming tracks within 60 s were recorded.

Hematoxylin and eosin (HE)/ Nissl staining

We fixed the perfused brain tissues in 4% paraformaldehyde solution for 24 h after collection.

Then, the brain tissues were dehydrated in alcohol, embedded in paraffin wax, and cut into 5 μm thick sections from the coronal plane. Dewaxed brain sections were then rehydrated, dyed, dehydrated, and rendered transparent according to the instructions provided in the HE staining kit and Nissl staining kit. Finally, the slides were observed under a light microscope (Olympus, Japan).

Bioinformatics analysis

To navigate the expression level of CD44 in diseased and normal tissues, two datasets (GSE122063 and GSE161355), both from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) were utilized in this study. The GSE122063 datasets were obtained from the GPL16699 platform comprising 8 VAD, 12 AD, and 11 control brain tissue for further analysis [42]. The GSE161355 datasets from the GPL570 platform comprised six T2D and five control brain tissue [43]. The probe identification numbers were converted into the official gene symbols according to the GPL16699 and GPL570 platforms. After log2 transformation and normalization, the “LIMMA” package [44] built in R software (version 4.3.1) was used to identify the differentially expressed genes (DEGs). The cutoffs were P<0.05 and false discovery rate (FDR)<0.05. When multiple probes corresponded to one gene, the average expression was taken. In the procession, we uploaded these genes to Hiplot to draw Wayne's diagram. Then, CD44 expression was extracted from geneMatrix files to analyze the differentially expressed levels in the two datasets. Next, using the “GO plot” package, we evaluated enriched biological processes (BPs), molecular functions (MFs), and cellular components (CCs). The thresholds for enrichment analysis were PvalueCutoff=0.05 and qvalueCutoff=0.05. Then, we uploaded target genes into the STRING database to predict the protein-protein interaction network.

Quantitative real-time-polymerase chain reaction (qRT-PCR) analysis

Total RNA was extracted from the frozen brain tissue using TRIzol reagent, reverse-transcribed to cDNA, and amplified with a commercial One-step gDNA Remover. The qRT-PCR analysis was conducted on a CFX Connect real-time PCR system (Bio-Rad, CA, USA) using cDNA, forward and reverse primers, and SYBR Green Supermix. The GAPDH gene was used as the internal control and to calculate the relative expression level of mRNA. Gene-specific primers were as follows: CD44, F: 5’-TGGCTCATCATCTTGGCATCT-3’ and R: 5’-TCCTGTCTTCCACCGTCCC-3’; GAPDH, F: 5’-CCTCGTCCCGTAGACAAAATG-3’ and R: 5’-TGAGGTCAATGAAGGGGTCGT-3’.

Western blot analysis

After treatment with specific experimental conditions, the total protein from the hippocampus tissue was extracted by RIPA lysis buffer (Servicebio, G2002) containing a protease inhibitor (Servicebio, G2006), phosphatase inhibitor (Servicebio, G2007), and 0.1 M phenylmethylsulfonyl fluoride (Beyotime, ST507). A liquid nitrogen grinder and ultrasonic grinding were used to lyse the tissues. We then collected the supernatants and determined the protein content using the BCA reagent (Beyotime, P0010). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking by 5% nonfat milk, the membranes were incubated with different primary antibodies: insulin receptor substrate-1 (IRS-1; CST,#2382,1:1000), phospho-IRS-1(Ser307)(ABclonal, AP0552, 1:800), PI3K(p110)(ABclonal, A22730, 1:800), CD44(CST,#3570,1:1000), AKT(CST,#9272,1:1000), phospho-AKT (S473) (CST, #4060,1:1000), GSK3β (Wanleibio, WL10456,1:500), and phospho-GSK3β (CST,#5558,1:1000), tau (Proteintech, 10274-1-AP, 1:2000), phospho-tau (Proteintech, 82568-1-RR1:2000) at 4°C for 12 h~18 h. At room temperature, membranes were incubated for 1 h with the secondary antibodies: anti-mouse (Proteintech, SA00001-1, 1: 5000), anti-rabbit (Proteintech, SA00001-2, 1:5000). As the internal control, GAPDH (ABclonal, AC002, 1:5000) was used. The immunocomplexes were finally observed with a UVP BioSpectrum 415 Imaging System (Upland, CA, USA).

Statistical analysis

The bands obtained in the western blotting assay were analyzed by Image J software for gray value, SPSS 26.0 software for statistical analysis, and GraphPad Prism 8.0 was used for plotting. One-way analysis of variance (ANOVA) was used to compare the differences in data, the Student–Newman–Keuls-q test was used for further two-by-two comparisons, and the least significant difference test was used to compare the differences between groups. The results of the measurement conforming to the normal distribution were expressed as the mean±standard error of the mean, and the difference of P<0.05 was considered to be statistically significant.

Results

Cross-analysis of the molecular links between type 2 diabetes and Alzheimer’s disease

Previous studies uncovered genes and signatures crosstalk linked these two diseases [45] [46] [47] [48]. Herein, DEGs between AD and control included 381 downregulated genes and 357 upregulated genes in the dataset GSE122063 ([Fig. 1a]). DEGs between T2D and control included 95 downregulated genes and 256 upregulated genes in the dataset GSE161355 ([Fig. 1a]). The common upregulated genes included SERPINA3, GEM, MAFF, DNAJB1, SPP1, HSPB1, GFAP, and CD44. The common downregulated genes were ADHFE1 and BDKRB1. Enrichment analysis showed that the functions of most DEGs were enriched in cell projection, extracellular exosome, inflammatory response metascape, etc. ([Fig. 1c]). Among these DEGs, CD44 was particularly tightly correlated with diabetes, insulin resistance and inflammatory response [40] [49] ([Fig. 1d]). Herein, we verified that CD44 was upregulated in both two datasets with P<0.05. We found a significant difference in CD44 expression between the control group and the disease group in the two datasets ([Fig. 1b]). In the GSE122063 dataset, the P value for CD44 expression was<0.001 in the temporal or frontal cortex when comparing AD and control groups. In the GSE161355 dataset, the P value of CD44 expression was<0.05, in the temporal cortex when comparing T2D and control groups ([Fig. 1b]). The Gene Ontology (GO) enrichment analysis of the common DEGs between AD and T2D revealed CD44, which is involved in cell projection, extracellular exosome, inflammatory response, and protein binding ([Table 1]).

Fig. 1 (a) The Wayne diagram shows differentially expressed genes (DEGs) in the microarray datasets GSE122063 and GSE161355. (b) CD44 expression in the temporal cortex in GSE122063 and GSE161355 datasets. *P<0.05, ***P<0.001, unpaired T-test. (c) Gene Ontology (GO) enrichment bubble diagram analysis of the common DEGs between Alzheimer’s disease and type 2 diabetes. (d) Protein-Protein Interaction network of relative protein in homo sapiens.

Table 1 Gene Ontology enrichment analysis of the common differentially expressed genes between Alzheimer’s disease and type 2 diabetes. GO enrichment analysis of the common DEGs between AD and T2D.

Category	Term	Description	P	Genes
Biological Processes	GO: 0006954	Inflammatory response	0.009462311	SERPINA3, SPP1, CD44
	GO: 0006986	Response to unfolded protein	0.020729416	DNAJB1, HSPB1
Cellular Components	GO: 0042995	Cell projection	0.001755779	SPP1, CD44, GFAP
	GO: 0070062	Extracellular exosome	0.003577379	DNAJB1, SERPINA3, SPP1, HSPB1, CD44
Molecular Functions	GO: 0044183	Protein binding involved in protein folding	0.018695455	DNAJB1, HSPB1
	GO: 0051082	Unfolded protein binding	0.047063495	DNAJB1, HSPB1
	GO: 0005178	Integrin binding	0.056947804	SPP1, GFAP
	GO: 0005515	Protein binding	0.059081429	DNAJB1, CD44, GEM, GFAP MAFF, SERPINA3, SPP1, HSPB1,

GO: Gene Ontology

Echinacoside could alleviate general health disorders and insulin resistance in diabetic mice

Changes in body weight and fasting plasma glucose of the three groups were examined fortnightly. The db/db+ECH and db/db groups exhibited significantly increased weight gain, greater than that of the db/m group at the beginning of diet treatment, and this significant difference was maintained throughout the remaining weeks ([Fig. 2a]). However, compared with the db/db group, the db/db+ECH group had a relatively lower body weight gain. Likewise, the FPG level of db/db mice increased significantly and fluctuated dramatically compared to that of the db/m group ([Fig. 2b]), and this level and fluctuation of FPG decreased as a result of ECH intervention compared to that of the db/db group. Following OGTT, the db/db group experienced a significant delay in glucose clearance ([Fig. 2d]). The AUC was significantly higher in db/db ([Fig. 2e]). ECH intervention significantly reduced AUC, and improved glucose clearance in db/db mice ([Fig. 2d-e]).

Fig. 2 Measurements of general health conditions and insulin resistance in db/m, db/db, and db/db-ECH groups. (a) Weight gain; circle=db/m group, rectangle=db/db group, triangle=db/db-ECH group. (b) FPG; circle=db/m group, rectangle=db/db group, triangle=db/db-ECH group. (c) Fasting insulin (FINS mIU/L), left column=db/m group (6.56±0.19), middle column=db/db group (22.09±0.48), right column=db/db-ECH group (8.56±0.54). (d) OGTT (mmol/L), circle=db/m group, rectangle=db/db group, triangle=db/db-ECH group. (e) Plasma glucose AUC of OGTT (*100 mmol/L*min), left column=db/m group (12.44±0.53), middle column=db/db group (36.93±0.53), right column=db/db-ECH group (21.00±1.26). (f) insulin resistance index, left column=db/m group (0.81±0.05), middle column=db/db group (3.21±0.06), right column=db/db-ECH group (1.44±0.11), (insulin resistance index HOMA-IR=FPG×FINS/22.5, natural logarithms are taken for statistical treatment). Compared with the db/db group, *P<0.05, **P<0.01,***P<0.001. Compared with the db/m group, ^### P<0.001. n=7 per all group. OGTT: Oral Glucose Tolerance Test; FPG: fasting plasma glucose; ECH: Echinacoside; AUC: area under the curve.

To clarify the effects of ECH on insulin sensitivity, we further evaluated the level of insulin and HOMA-IR in each group at the end of the test. Insulin content in the db/db mice (22.09±1.26 mIU/L) was distinctly higher than that of the control group (6.57±0.51 mIU/L), and ECH intervention significantly decreased insulin levels ([Fig. 2c]). HOMA-IR is another reliable indicator for insulin resistance. HOMA-IR was significantly enhanced in the db/db group compared with the db/m group ([Fig. 2f]), indicating serious insulin resistance. After the ECH intervention, the HOMA-IR of the db/m group was notably diminished compared to that of the db/db group ([Fig. 2f]).

Partial restoration of the cognitive impairment in diabetic mice after echinacoside treatment

Morris water maze test, as a widely used behavioral experiment reflecting cognitive ability, was conducted to examine the spatial learning and memory ability of mice. The escape latency of the db/db group was significantly longer than that of the db/m group and db/db-ECH group during 5 days of training ([Fig. 3a]). Overall, the escape latency on day 5 of db/db-ECH (21.60±1.89) group was significantly lower than that of the db/db group (38.73±4.52), indicating that ECH improved the cognitive function of mice with T2D ([Fig. 3b]). Similarly, compared with the db/m group, the db/db group showed cognitive impairment, with a significantly lower value of target platform crossings, a short time in platform area and target quadrant in the probe trial ([Fig. 3c-e]). After the ECH intervention, the platform crossing ([Fig. 3c]), the time in platform ([Fig. 3d]), and the platform quadrants ([Fig. 3e]) of the db/db group increased significantly. The swimming track of the db/db group tended to be marginal, while the db/db-ECH group showed a more active way of exploration ([Fig. 3f]). These data indicated that ECH partially restored the learning and memory impairment of diabetic mice.

Fig. 3 Morris water maze assessments in db/m, db/db, and db/db-ECH group. (a) Escape latency during five days of training (red=db/m group, yellow=db/db group, purple=db/db-ECH group). (b) The escape latency on day 5, the left column=db/m group (20.29±3.02), the middle column=db/db group (38.73±4.52), the right column=db/db-ECH group (21.60±1.89). (c) Numbers of target platform crossings, db/m group (1.86±0.12), db/db group (0.57±0.17), db/db-ECH group (1.86±0.35). (d) time in platform area, db/m group (3.44±0.39), db/db group (0.6±0.19), db/db-ECH group (3.07±0.40). (e) Time in target quadrant, db/m group (31.24±1.78), db/db group (17.14±1.05), db/db-ECH group (27.12±1.96). (f) Representative swimming tracks. Mean±standard error of the mean. n=7 per group. *P<0.05, **P<0.01, ***P<0.001, one-way analysis of variance. Compared with db/db group, *P<0.05, **P<0.01, ***P<0.001. Compared with db/m group, ^## P<0.01. n=7 per all group. ECH: Echinacoside.

Echinacoside could ameliorate the histomorphologic damage of the hippocampus in diabetic mice

For exploring the effects of ECH on brain damage, the cell arrangement and number of neurons in the hippocampus of diabetic mice were examined by HE and Nissl staining. The hippocampal regions in the db/m group presented regular cell arrangement in HE staining. In the db/db group, a disordered arrangement and nuclei pyknosis of neurons were observed in the dentate gyrus (DG), field CA3 of the hippocampus (CA3), and field CA1 of the hippocampus (CA1) regions ([Fig. 4a]). After ECH was gavaged, these damages were alleviated ([Fig. 4a]). The number of neurons in the control and treatment groups was observed after Nissl staining ([Fig. 4b]). In the db/db group, the number in the DG and CA3 regions was remarkably reduced compared with those in the db/m group ([Fig. 4c]). After being gavaged with ECH, the number of surviving neurons in hippocampal DG and CA3 areas of the ECH group was notably enhanced ([Fig. 4b]). These results displayed that ECH prevented the loss of neurons in the hippocampus of diabetic mice.

Fig. 4 (a) The HE staining diagram of the hippocampus in mice from the db/m, db/db, and db/db-ECH group. DG: dentate gyrus, CA3: field CA3 of the hippocampus, CA1: field CA1 of the hippocampus (magnification:×200, bar=50 μm). (b) The Nissl staining image of the hippocampus in mice from the db/m, db/db, and db/db-ECH groups (magnification:×400, bar=20 μm). (c) Quantitative estimation of the neuron number in the hippocampus by Nissle staining, the left column=db/m group, the middle column=db/db group, the right column=db/db-ECH group. Mean±standard error of the mean. n=7 per group. *P<0.05, **P<0.01, ***P<0.001, one-way analysis of variance. HE: hematoxylin and eosin; ECH: Echinacoside.

Reduced mRNA and protein expression of CD44 in diabetic mice after echinacoside treatment

To verify the upregulated expression of CD44 in T2DM and the potential link to ECH, the expression levels of mRNA and protein in the db/db mice were evaluated by qRT-PCR and western blotting. As shown in [Fig. 5], CD44 mRNA and protein expressions in the db/db group were remarkably decreased compared with those in the db/m group (P<0.01, P<0.05). However, the treatment with ECH remarkably restored the mRNA and protein expressions compared with those in the db/m group.

Fig. 5 (a) Determination of CD44 mRNA expression by quantitative real-time-polymerase chain reaction. (b) Determination of CD44 protein expression by western blotting. Mean±standard error of the mean. *P<0.05, **P<0.01, one-way analysis of variance.

Echinacoside affected the phosphorylation of IRS-1/PI3K/AKT/GSK-3β and tau in diabetic mice

Hyperphosphorylation of tau(p-tau) aggregation in neurofibrillary tangles (NFTs) is closely associated with cognitive decline in neurodegeneration disease [34] [35]. The balance of p-tau/tau is regulated by GSK3β, which is negatively regulated by phosphorylation at ser9 [36]. Western blotting to determine the expression of GSK-3β, p-GSK-3β(Ser9), tau, and p-tau(S202/T205) protein among different groups ([Fig.6a-b]), revealed no significant difference among the three groups in terms of the expression of GSK3β and tau proteins; however, the content of phosphorylated proteins varied. As shown in [Fig.6a-b], the ratio of p-GSK-3β(Ser9)/GSK-3β in the diabetic group was lower than that in the db/m group (P<0.05), but the ratio of p-tau(S202/T205)/tau protein was higher. ECH administration enhanced the ratio of p-GSK-3β(Ser9)/GSK-3β and decreased the ratio of p-tau(S202/T205)/ tau compared to that of the diabetic group (P<0.05). Less tau phosphorylation is associated with reduced pathological alterations. ECH-induced decrease in relative p-tau(S202/T205) level indicated its effect on rescuing detrimental changes in the brain of diabetic mice.

Fig. 6 (a) The expression of GSK-3β, phosphorylated (p)-GSK-3β(Ser9), tau and p-tau(Ser202/Thr205) in brain tissues from the db/m, db/db, and db/db-ECH groups. (b) Quantitative assessment of these proteins (mean±SEM; *P<0.05 through one-way ANOVA). (c) The expression of IRS, p-IRS(S307), p-PI3K(110), pAKT(S473), and AKT in brain tissues from the db/m, db/db, and db/db-ECH group. (d)Quantitative assessment of these proteins. Mean±SEM. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA. IRS: insulin receptor substrate-1; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B; SEM: standard error of the mean; ANOVA: analysis of variance.

Gsk3β is mainly regulated by the IRS-1/PI3K/AKT insulin signaling pathway [36] [38]. The mode of action of ECH on GSK-3β phosphorylation was investigated by performing western blotting to evaluate the levels of IRS-1/PI3K/AKT pathway proteins in the three groups. As shown in [Fig.6c-d], the levels of IRS-1 and AKT proteins in the db/db mice had not changed significantly compared with those in the db/m mice group. However, changes were observed in phosphorylated IRS-1, PI3K, and AKT proteins. The expression of p-IRS1(S307)/IRS1 in the diabetic group was higher than that in the db/m group (P<0.01), while that of p-PI3K(110) (P<0.01), and p-AKT(S347)/AKT was lower (P<0.001). Treatment with ECH caused a remarkable restoration of the expression of these proteins, which was in contrast to those in the diabetic group. The expression of P-IRS1(S307)/IRS1 was decreased (P<0.05), but that of p-PI3K (110)(P<0.05) and p-AKT(S473)/AKT(P<0.01) were enhanced.

Discussion

In recent years, T2D-induced cognitive dysfunction is gaining attention; some researchers refer to AD as type 3 diabetes mellitus or cerebral diabetes mellitus, which reinforces the strong link between T2D-induced cognitive dysfunction and AD [13]. Studies have shown that chronic inflammation, Aβ deposition, p-tau, and some cell signaling pathways play important roles in the progression of both T2D and AD. In this study, we demonstrated that ECH ameliorates T2D-induced cognitive dysfunction in db/db mice. Our study shows that ECH ameliorates the histomorphologic damage of the hippocampus and improves cognitive and learning functions in diabetic mice [50]. Moreover, ECH also reduced the mRNA and protein expression of CD44. Subsequently, our mechanistic experiments verified that ECH affected the phosphorylation of GSK-3β and tau, as well as the IRS-1/PI3K/AKT insulin signaling pathway in diabetic mice.

Herbal medicines are increasingly valued in the treatment of diabetes. ECH, a phenylethanol glycoside, as the most biologically active compound of C. tubulosa, has been reported to benefit diabetic cardiomyopathy through p53/p38 mitogen-activated protein kinase and peroxisome proliferator-activated receptorα/mast cell protease-1 signaling, inhibiting kidney fibrosis via the transforming growth factor-β1/Smad pathway and benefit hepatic steatosis by the sterol regulatory element-binding protein1c/ fatty acid synthase pathway [15] [21] [51], but the role of ECH in diabetic encephalopathy has not yet been elucidated. Our study demonstrates that ECH can ameliorate hippocampal damage in diabetic encephalopathy and may provide some new options for the treatment of patients with diabetic encephalopathy.

IRS1 is a substrate of the islet receptor tyrosine kinase that becomes activated and plays an important role in insulin signaling [11]. Tyrosine phosphorylation of IRS exposes binding sites for numerous signaling chaperones to bind to, including PI3K/Akt, which affects insulin function. In recent years, It has also been found that the PI3K/Akt pathway can induce hippocampal damage and neuroinflammation, and promote tau phosphorylation through GSK-3β, leading to cognitive dysfunction [49] [50] [52]. In the current study, we found that ECH can ameliorate hippocampal damage and tau hyperphosphorylation through the IRS1/PI3K/Akt pathway. These findings may provide a better understanding of the pathogenesis of diabetic encephalopathy.

CD44, a cell surface glycoprotein, is highly expressed in pancreatic islets and renal cortex of diabetic mice and has been shown to promote tau accumulation. CD44 has also been shown to influence the progression of hepatocellular carcinoma and cholangiocellular carcinoma through the Akt pathway [40] [41] [53]. Our study found elevated CD44 levels in the brains of db/db mice, with a significant decrease after ECH treatment.

The limitation of our study is that we did not elucidate the specific mechanism by which ECH regulates the IRS/PI3K/Akt pathway and did not use the classical Akt pathway inhibitors to compare the effect of ECH. We will continue to focus on these issues and perform further studies. Additionally, we subjected mice to an 8-h fasting period every 2 weeks to measure FPG. Although we measured FPG before the mice were fed each morning, we do not know if this may have caused metabolic and stress challenges in mice.

In conclusion, our study identifies the potential of ECH as a drug that can improve diabetic encephalopathy via CD44 and the IRS1/PI3K/Akt pathway. This finding provides a new option for the treatment of patients with diabetic encephalopathy.

Author Contributions

RH conceived and designed the experiments. JY, JZ, CY, JL, and FQ, YY, and NY performed the experiments. FQ and YY analyzed data and contributed reagents, materials, and analysis tools. FQ interpreted the results and wrote the paper. All authors made contributions to the article and approved the final version for submission.

Declarations

Ethics approval and consent to participate

The approval for the experimental procedures was obtained from the Institutional Animal Care and Use Committee of Renmin Hospital, Wuhan University (Issue number: WDRM20190517).

Available of data and materials

The Gene Expression Omnibus database was accessed through https://www.ncbi.nlm.nih.gov/geo/. The Hiplot data are available on https://hiplot.com.cn/cloud-tool/drawing-tool/list). The Enrichment analysis data are available on http://metascape.org/gp/index.html#/main/step1). The PPI network data were obtained from https://string-db.org/.

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