Xianan Dong,Liangliang Kong,Lei Huang,Yong Su,Xuewang Li,Liu Yang,Pengmin Ji,Weiping Li,Weizu Li
Ginsenoside Rg1 treatment protects against cognitive dysfunction via inhibiting PLC–CN–NFAT1 signaling in T2DM mice
Type II Diabetes Mellitus,Cognitive dysfunction,Ginsenoside Rg1,Calcium homeostasis,Phospholipase C
Background As a complication of Type II Diabetes Mellitus (T2DM), the etiology, pathogenesis, and treatment of cognitive dysfunction are still undefined. Recent studies demonstrated that Ginsenoside Rg1 (Rg1) has promising neuroprotective properties, but the effect and mechanism in diabetes-associated cognitive dysfunction (DACD) deserve further investigation. Methods After establishing the T2DM model with a high-fat diet and STZ intraperitoneal injection, Rg1 was given for 8 weeks. The behavior alterations and neuronal lesions were judged using the open field test (OFT) and Morris water maze (MWM), as well as HE and Nissl staining. The protein or mRNA changes of NOX2, p-PLC, TRPC6, CN, NFAT1, APP, BACE1, NCSTN, and Aβ1-42 were investigated by immunoblot, immunofluorescence or qPCR. Commercial kits were used to evaluate the levels of IP3, DAG, and calcium ion (Ca2+) in brain tissues. Results Rg1 therapy improved memory impairment and neuronal injury, decreased ROS, IP3, and DAG levels to revert Ca2+ overload, downregulated the expressions of p-PLC, TRPC6, CN, and NFAT1 nuclear translocation, and alleviated Aβ deposition in T2DM mice. In addition, Rg1 therapy elevated the expression of PSD95 and SYN in T2DM mice, which in turn improved synaptic dysfunction. Conclusions Rg1 therapy may improve neuronal injury and DACD via mediating PLC–CN–NFAT1 signal pathway to reduce Aβ generation in T2DM mice.
Ginsenoside Rg1 treatment protects against cognitive dysfunction via inhibiting PLC–CN–NFAT1 signaling in T2DM mice

As a complication of Type II Diabetes Mellitus (T2DM), the etiology, pathogenesis, and treatment of cognitive dysfunction are still undefined. Recent studies demonstrated that Ginsenoside Rg1 (Rg1) has promising neuroprotective properties, but the effect and mechanism in diabetes-associated cognitive dysfunction (DACD) deserve further investigation.

After establishing the T2DM model with a high-fat diet and STZ intraperitoneal injection, Rg1 was given for 8 weeks. The behavior alterations and neuronal lesions were judged using the open field test (OFT) and Morris water maze (MWM), as well as HE and Nissl staining. The protein or mRNA changes of NOX2, p-PLC, TRPC6, CN, NFAT1, APP, BACE1, NCSTN, and Aβ1-42 were investigated by immunoblot, immunofluorescence or qPCR. Commercial kits were used to evaluate the levels of IP3, DAG, and calcium ion (Ca2+) in brain tissues.

Rg1 therapy improved memory impairment and neuronal injury, decreased ROS, IP3, and DAG levels to revert Ca2+ overload, downregulated the expressions of p-PLC, TRPC6, CN, and NFAT1 nuclear translocation, and alleviated Aβ deposition in T2DM mice. In addition, Rg1 therapy elevated the expression of PSD95 and SYN in T2DM mice, which in turn improved synaptic dysfunction.

Rg1 therapy may improve neuronal injury and DACD via mediating PLC–CN–NFAT1 signal pathway to reduce Aβ generation in T2DM mice.

With social development, lifestyle changes, and the aging population, the number of patients with diabetes mellitus (DM) is increasing year by year [1]. The link between DM and cognitive dysfunction has been proposed as early as 1922 [2]. Furthermore, epidemiological, animal and clinical researches further show a tight internal relationship between DM and cognitive dysfunction [3]. According to growing evidence, DM is an independent hazard factor for cognitive dysfunction, which can lead to cognitive deterioration, mild cognitive dysfunction, and dementia [4,5]. Unfortunately, there was still no efficient therapy to reverse diabetes-associated cognitive dysfunction (DACD) at present. Therefore, developing novel therapeutic strategies and drugs for DACD are urgently needed. DACD appears to be caused by a combination of causes [6]. To begin with, chronic hyperglycemia and hyperlipidemia are the primary causes of the occurrence and progression of DACD [7,8]. Moreover, disturbed lipid and protein metabolism, as well as abnormal insulin signaling, contribute to neuronal lesions and cognitive dysfunction in DM [9,10]. Recently, it has been reported that neuronal calcium overload was closely related to diabetic neurological disease and neurodegenerative diseases [11]. Phospholipase C (PLC), which can cleave inositol diphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3), is involved in lipid metabolism and increases IP3 and DAG-induced endoplasmic reticulum calcium (Ca2+) release and extracellular Ca2+ entry [12]. As a nonselective cation channel, transient receptor potential channel 6 (TRPC6) have higher Ca2+ permeability than other cations that can be activated by DAG and responds to PLC [13]. Calcineurin (CN) is a Ca2+-dependent enzyme that is regulated by intracellular Ca2+ levels ([Ca2+]i) [14]. CN response to increased [Ca2+]i, which then increases the nuclear factor of activated T-cells 1 (NFAT1) to regulate target gene transcription [15]. During the initial stages of Alzheimer's disease (AD), NFAT1 has been shown to be activated in the hippocampus [16]. Recent studies have shown that in the presence of Aβ oligomers, knocking down Nav1.6 inhibits calcium overload, consequently increasing inactive NFAT1 levels and thus reducing BACE1 transcription [17], suggesting that this mechanism may closely contribute to regulation of Aβ deposition in APP/PS1 double-transgenic mice. Moreover, resveratrol, a polyphenol found in grapes, has been shown to inhibit TRPC6 and CN, therefore protecting against ischemic nerve damage [18]. However, it's still unclear whether the PLC–CN–NFAT1 signaling involves in DACD. With the development of drugs, bits of anti-diabetic drugs have shown a delayed effect on DACD [19], but many of these drugs have unfavorable adverse reactions. Hence, greater resources should be dedicated to researching the possible potential dangers, pathogenesis, and therapeutic drugs for DACD. Ginsenoside Rg1 (Rg1) is a substance of ginseng that has shown excellent antioxidant and anti-aging properties [20,21]. Furthermore, recent studies suggest that Rg1 can alleviate insulin resistance and regulate glucose metabolism, indicating Rg1's potential role in type II diabetes mellitus (T2DM) [22]. Hence, Rg1 is likely to be a pharmacological candidate for preventing and treating DACD. This study was performed to test our hypothesis that Rg1 treatment may reduce NADPH oxidase 2 (NOX2)-derived ROS accumulation and inhibit PLC-NFAT1-BACE1 signaling, thus improving neuronal lesions and cognitive dysfunction in T2DM mice. This research might provide a novel treatment strategy for T2DM-related cognitive dysfunction.

The male C57/BL mice (18–21 g) were purchased from the Anhui Medical University Laboratory Animals Center and housed in standard laboratory conditions. After adaptive feeding, mice were separated into seven groups (n = 12) at random: control group, high-fat diet (HFD) group, HFD + streptozocin (HFD + STZ) group, Rg1 (1, 5 or 10 mg/kg) groups, and metformin (200 mg/kg) group. The mice in the control group were allowed free access to standard chow and water. The remaining mice were given HFD (Xietong Bio-engineering, XTHF45-1) for 8 weeks, then STZ (110 mg/kg, Solarbio, S8050) injections were given intraperitoneally except for HFD group. The concentration (≥ 16.8 mM) of fasting blood glucose (FBG) after 72 hours was measured to estimate whether T2DM models were successfully built. Then the Rg1 (content > 98%, Desite, DR0009) and metformin (Chiatai Tianqing Pharmaceutical) were treated by gavage delivery for 8 weeks. The timeline representing the experimental design was shown in (Supplementary Fig. S1A). The animal experiment was performed under the supervision of the Animal Ethics Committee of Anhui Medical University.

The OFT was carried out exactly as the previous study stated [23]. The changes in behavior were determined at 0-week (before the high-fat diet), 12-week (4 weeks after Rg1 treatment), and 16-week (8 weeks after Rg1 treatment). The changes in the number of stand up and line crossing, moving distance and speed were calculated to indicate the motor activity and exploratory behavior of T2DM mice.

Consistent with our previous study used protocol, the experiment included four days of training and one day of testing [24]. The mean escape latency (MEL, s) was recorded during the period of 1-4 days. The platform was hidden on the fifth day, and the activity was recorded for one minute to estimate the changes in spatial learning and memory of T2DM mice.

HE and Nissl staining were carried out as a previous study stated [25]. The neuronal morphologic and Nissl bodies changes in cortical, hippocampal CA1 and CA3 zones were examined by an automatic digital pathology scanning device (Pannoramic Midi II, 3DHISTECH). The mean density of Nissl staining was calculated using Image-Pro Plus 6.0 software to determine the level of Nissl bodies.

Tissue slices were covered with primary antibodies of PSD95 (1:200, Servicebio, GB11277) or Aβ1-42 (1:100, Bioss, bs-23379R) overnight at 4°C and placed in a concealed box with water on the bottom. The next day, slices were bath in PBS and then incubated for 1 hour at room temperature with a rhodamine-labeled secondary antibody (1:200, ZSGB-BIO, ZF-0316). The density of tagged protein was computed using Image-Pro Plus 6.0 software after the data was gathered.

Briefly, the DHE (Beyotime, S0063) was injected (100 μM, 0.1 ml/10g) for 30 minutes from the tail vein. When the embedding agent OCT (Sakura, Torrance, CA) solidifies, the brain was sliced with a freezing microtome into 10 μm sections. Then the slices were covered with DAPI for 10 minutes for nuclear dye and were examined by an automatic digital pathology scanning device. The density of red fluorescent to indicate ROS generation was also computed using Image-Pro Plus 6.0 software.

The product manuals were followed for RNA extraction (Beyotime, R0027), then used for the reverse transcription (Takara, RR036A). The qPCR kit (Takara, RR430A) was used to perform real-time experiments and the 2−ΔΔCt method was used to analyze transcript levels. Supplementary Table S1 lists the primer sequences used in the qPCR experiments.

A commercial kit (Beyotime, P0028) was used to separate cytoplasmic and nuclear proteins in brain. The equivalent amount of proteins was separated through SDS-PAGE gel when protein concentration was quantified by BCA. After electrophoresis, proteins were transferred to the PVDF membrane by wet transfer and closed by skim milk. The PVDF membranes were exposed with ECL after incubation with primary antibody (4°C, overnight, Supplementary Table S2) and secondary antibody (RT, 1 hour). The bands were obtained by Imaging system (ChemiDoc, Bio-Rad, USA) and quantified by Image J software.

Concentrations of IP3 and DAG in the brain tissue were detected by ELISA (Pyram Biotechnology, PM105479 & PM104601) under the manufacturer's instructions. Similarly, Calcium Colorimetric Assay Kit (Beyotime, S1063) was utilized to evaluate the relative levels of Ca2+ in the brain tissue homogenate.

The data were demonstrated as mean ± SD. All data was processed by GraphPad Prism 8 software package, and P < 0.05 was regarded as significantly different. Two-way ANOVA with repeated measures was performed to analyze the data of body weight, FBG, food intake, water intake, OFT and MWM training trials. Other results were processed by one-way ANOVA accompanied by Tukey's test for multiple group comparisons.

The changes in body weight were detected every two weeks. The FBG, the food, and the water intake were measured every two weeks after modeling. During 0-8 weeks, all animals' body weight gradually increased but declined at 8-16 weeks after modeling except for control and HFD groups. Rg1 (5 and 10 mg/kg), and metformin therapy improved the body weight loss at 14 and 16 weeks when compared with HFD + STZ group (Supplementary Fig. S1B). After modeling, the FBG levels were significantly increased, while Rg1 (5 and 10 mg/kg) and metformin therapy ameliorated FBG levels at 14 and 16 weeks compared with HFD + STZ group (Supplementary Fig. S1C). Meanwhile, the food and water intake were significantly increased at 12-16 weeks in HFD + STZ group, and Rg1 (5 and 10 mg/kg) therapy could markedly decrease the water intake (Supplementary Fig. S1D & E). These results proved that Rg1 possesses a great ability to improve the diabetic symptoms of T2DM mice. We also detected the motor function by OFT at 0, 12, and 16 weeks. The results showed no differences in behavioral performance at 0-week before HFD treatment (Fig. 1B–E). In contrast with the control group at 12 and 16 weeks, the moving distance (m) and moving speed (m/s) had no significant changes, and the number of stand up and line crossing had a significant decrease at 16 weeks in HFD group, but they were all seriously declined in HFD + STZ group mice (Fig. 1B–E). Rg1 (5 and 10 mg/kg), and metformin therapy considerably enhanced motor function in T2DM mice at 12 and 16 weeks compared with HFD + STZ group (Fig. 1B–E). According to these findings, Rg1 therapy could improve the motor and exploratory behavior of T2DM mice. We further detected learning and memory using MWM at 16 weeks. The results demonstrated that the mean escape latency (MEL, s) in HFD group have no difference with control group, but it was significantly increased in HFD + STZ group on days 2-4 in training trials (Fig. 1G). Rg1 (10 mg/kg) and metformin therapy could reduce the MEL on days 3 and 4 as compared to HFD + STZ group (Fig. 1G). The results of space exploration on the fifth day demonstrated that HFD + STZ treatment significantly decreased the swimming distance (m), swimming time in the platform (STP, s), and the number of crossing platform (NCP), and prolonged the latency of first entry to platform (LFP, s) (Fig. 1H–K). Rg1 (5 and 10 mg/kg), and metformin therapy impressively corrected these alterations in T2DM mice as compared to HFD + STZ group (Fig. 1H–K). All of these findings revealed that Rg1 medication could protect T2DM mice from learning and memory dysfunction, and HFD treatment had no influence on cognition.

We firstly examined neuronal morphologic and Nissl bodies change by HE and Nissl staining. The results of HE revealed that neither the control nor HFD groups had few neuronal lesions. While in the HFD + STZ group, more neurons were irregular and nuclear pyknosis. Rg1 and metformin therapy dramatically improved neuronal lesions in T2DM mice as compared to the HFD + STZ group, (supplementary Fig. S2). The Nissl staining also revealed that both the control and HFD groups had sufficient Nissl bodies. However, the Nissl bodies were considerably diminished in cortical and hippocampal neurons of HFD + STZ group mice (supplementary Fig. S3) and were considerably improved by Rg1 and metformin medication (supplementary Fig. S3B-D). We also observed the expression of PSD95 and SYN, two proteins involved in synaptic plasticity [26]. In HFD + STZ group mice, the expression of PSD95 in the cortical and hippocampal zones was generally lower than that in control group according to immunofluorescent staining findings (Fig. 2A and B). Compared to HFD + STZ group, Rg1 and metformin therapy improved the reduction of PSD95 in T2DM mice, particularly in Rg1 (10 mg/kg) dosage (Fig. 2A and B). The immunoblot data were consistent with the immunofluorescent staining, Rg1 and metformin therapy dramatically restored PSD95 and SYN reduction in the brain of T2DM mice (Fig. 2C–E). Moreover, the results also indicated that HFD treatment has no impact on the expressions of PSD95 and SYN (Fig. 2A–E). All of these findings demonstrated that Rg1 therapy can attenuate neuronal lesions of T2DM mice.

We further investigated the Aβ generation in the brain of T2DM mice. The immunoblot findings revealed that the expression levels of amyloid precursor protein (APP), β-secretase (BACE1), γ-secretase (NCSTN), β-carboxy-terminal fragments (CTF-β), Aβ1-42, and p-Tau/Tau were considerably increased in HFD + STZ group compared to control group. Rg1 (5, 10 mg/kg) and metformin therapy considerably downregulated these proteins generation as compared to HFD + STZ group (Fig. 3A–C & G-I). The qPCR results of APP, BACE1 and NCSTN were consistent with the immunoblot results (Fig. 3D–F). To further assess the function of Rg1 medication on Aβ deposition, immunofluorescent staining was used to evaluate the expression of Aβ1-42 (supplementary Fig. S4A). When compared to HFD + STZ group, the results confirmed that Rg1 and metformin medication could ameliorate Aβ1-42 generation in the brain of T2DM mice (supplementary Fig. S4B-D). Moreover, the results also demonstrated that HFD has no impact on the generation of Aβ. Overall, the data above showed that Rg1 could alleviate Aβ production in the brain of T2DM mice by modulating APP processing.

We firstly applied DHE staining to observe the generation of ROS in the brain. The data showed that there was almost no ROS overproduction in the cortical and hippocampal zones of the control and HFD groups (Fig. 4A–D). In contrast, the generation of ROS in HFD + STZ group was markedly elevated than that in control group (Fig. 4A–D). As compared to HFD + STZ group, Rg1 and metformin treatment lowered the accumulation of ROS in the brain of T2DM mice, particularly in the Rg1 (10 mg/kg) group. NOX2 has been reported to be one of the primary provenances of ROS production within brain neurons [27]. We further determined the expressions of NOX2-related components p47phox, p22phox, and NOX2 by immunoblot and qPCR. The expressions of NOX2-related components p47phox, p22phox, and NOX2 were dramatically enhanced in the brains of HFD + STZ group mice from Immunoblot data (Fig. 4E–H). When compared to HFD + STZ group, Rg1 and metformin treatment downregulated the expressions of NOX2-related components p47phox, p22phox, and NOX2, particularly in Rg1 (10 mg/kg) group (Fig. 4E–H). Our findings of qPCR were similar to immunoblot, the levels of p47phox and NOX2 mRNA were remarkably increased in HFD + STZ group, and markedly decreased by Rg1 and metformin (Fig. 4I–J). Moreover, the results also indicated that HFD has no significant impact on ROS accumulation and NOX2-related proteins expression. Overall, our results indicated that NOX2-mediated ROS generation is involved in the onset of cognitive dysfunction in T2DM mice, and Rg1 can improve cognitive impairment by reducing NOX2-mediated ROS generation.

PLC hydrolyzes PIP2 to generate IP3 and DAG, both of which are involved in Ca2+ homeostasis [28]. Therefore, we examined the levels of IP3, DAG and Ca2+ in the brain homogenates. According to our findings, the concentration of IP3, DAG, and Ca2+ was dramatically increased in HFD + STZ group mice and decreased after Rg1 and metformin treatment (Fig. 5D–F). The immunoblot results also indicated that the expressions of p-PLC, CN, TRPC6 and NFAT1 in the brains of HFD + STZ group were significantly increased, and were reduced by Rg1 and metformin treatment (Fig. 5A–C, G-L). The mRNA levels of CN and TRPC6 were consistent with the immunoblot results (Fig. 5I and J). Meanwhile, HFD treatment has no significant influence on PLC–CN–NFAT1 signaling in the brain (Fig. 5A-L). These data revealed that Rg1 therapy might alleviate neuronal injury and Aβ generation by regulating the PLC–CN–NFAT1 signal pathway in T2DM mice.

Increasing studies show that diabetes-associated cognitive dysfunction (DACD) is an important complication of T2DM [29]. However, the pathogenesis of DACD is still not fully understood, and rare clinical interventions are accessible to improve the sickness. Rg1, a ginseng extract, has anti-aging, neuroprotective and cognitive-improving properties [20,30]. The purpose of our study was to observe whether Rg1 therapy improves cognitive dysfunction and neuronal lesion in T2DM mice, and if so, what the mechanisms are. Our data demonstrated that Rg1 treatment ameliorated cognitive dysfunction and neuronal lesions, decreased ROS accumulation and Aβ generation, and inhibited PLC–CN–NFAT1 signaling in T2DM mice, suggesting that Rg1 may be a promising agent for the therapy of DACD. T2DM is a global health issue that can lead to several complications, one of which is DACD, which affects approximately 60-70 percent of people and manifests as a decline in learning and memory abilities, deficiency of concentration and analytical capability and so on [31,32]. Currently, DACD has received lots of attention in the therapy of diabetic complications because of its negative effects, which include neuropathy and cognitive dysfunction [33,34]. Similar to earlier reports [35], our study also proved that T2DM mice exhibited significant cognitive dysfunction in behavior tests. The results of HE and Nissl staining demonstrated severe neuronic injuries in the cortical and hippocampal zones of T2DM mice. Furthermore, synaptic impairment was considered an important representation in the development of DACD [36]. We further confirmed that the expressions of both SYN and PSD95 were decreased in the cortical and hippocampal zones in T2DM mice, but were considerably restored by Rg1 and metformin medication. And Rg1 and metformin treatment significantly ameliorated motor activity and spatial learning and memory dysfunction and attenuated neuronic lesions induced by T2DM. These findings show that Rg1 therapy protects against neuronal damage and cognitive dysfunction in T2MD mice. Currently, oxidative stress is thought to play an important role in the process of DACD and AD onset and progression. The overproduction of ROS can cause neuronal injury, resulting in Aβ overproduction and synaptic dysfunction [37]. NOX2 is a key generator of ROS in cerebral neurons and involved in neurodegenerative disorders [38]. It has been reported that NOX2 activity is linked to aging-associated neuronic injury and cognitive dysfunction [39]. Our prior findings have proved that NOX4 activation is an important mechanism in diabetic nephropathy [40]. Our latest research also suggested that Rg1 alleviates neuronic injury and Aβ deposition through decreasing NOX2 expression in APP/PS1 double-transgenic mice [41]. However, it is still unknown whether NOX2 involves in DACD. In the current investigation, we discovered that Rg1 remarkably reduced ROS generation in the cortical and hippocampal zones in T2DM mice. Furthermore, Rg1 therapy lowered the protein and mRNA expressions of p47phox p22phox, and NOX2, which is the key enzyme in neuronal ROS generation [27]. Our findings revealed the importance of NOX2-mediated ROS during the progression of DACD, which may relevant to the protective effect of Rg1 against DACD. However, how ROS accumulation causes Aβ overproduction drew our attention. Numerous reports indicated that calcium (Ca2+) dysregulation was related to the pathogenesis of various neurological sicknesses such as AD [42,43]. We speculated that Ca2+ overload may participate in DACD. Our data proved that the levels of Ca2+ in the brain were abnormally elevated in T2DM mice and ameliorated by Rg1 treatment, suggesting the involvement of calcium overload in DACD. ROS accumulation has been reported to play an important function in regulating Ca2+ influx and Ca2+ storage by activating PLC [44]. PLC is a widespread intracellular enzyme that catalyzes PIP2 conversion to DAG and IP3 [45]. IP3R located on the ER membrane can be activated by IP3 causing Ca2+ to be released rapidly from the endoplasmic reticulum to the cytoplasm [45], while DAG can activate TRPC6 inducing extracellular Ca2+ influx [46]. TRPC6 is a Ca2+-permeable non-selective cation channel with six-fold higher permeability to Ca2+ than to Na+, it is suspected to be implicated in the pathophysiology of DM [47,48]. Our previous study also indicated that upregulation of TRPC6 involves in renal fibrosis in diabetic nephropathy [49]. In our findings, the levels of DAG, IP3 and Ca2+ markedly increased in T2DM mice brain tissues and Rg1 treatment could decrease the concentration of DAG, IP3 and Ca2+. Further study indicated that Rg1 therapy considerably lowered the enhanced expressions of p-PLC and TRPC6 in T2DM mice brain tissues. These findings implied that Rg1 may ameliorate neuronal Ca2+ overload by inhibiting PLC-TRPC6 signaling in T2DM mice. Recent research reported that CN, a Ca2+-dependent protein phosphatase, is participated in many signaling pathways in cells. The nuclear factor of activated T cells (NFAT) can be activated by CN, which regulates gene transcription and leads to Aβ generation [50,51]. For instance, it has been reported that NFAT1 was significantly activated in Tg2576 mouse brains, and Ca2+ overload could activate CN-NFAT1 signaling and promote BACE1 gene expression by directly binding to the BACE1 promoter region [52,53]. In addition, NFAT plays a role in adipogenesis and glucose/insulin homeostasis, while hyperglycemia significantly up-regulated Ca2+/CN/NFAT4 axis [54,55]. These results implied that calcium homeostasis and the related CN/NFAT signaling pathway play a key role in diabetes-related complications. In the current study, we found that Rg1 therapy ameliorated calcium overload and inhibited the expressions of CN and NFAT1 and nuclear translocation of NFAT1 in T2DM mice. Based on previous reports and our existing results, we deduced that Rg1 exerts therapeutic effects through the CN/NFAT1 signaling pathway. Accumulating evidence has suggested diabetes as an independent risk factor for AD. Existing epidemiological evidence also suggests that T2DM elevates the relative risk of dementia and morbidity of AD [31]. Although the pathogenesis of DACD is yet unclear, it's worth noting that T2DM and AD share a lot of similarities [56]. For instance, growing evidence suggests that T2DM patients have learning and memory impairments, as well as increased Aβ accumulation and neurofibrillary tangles due to Tau protein hyperphosphorylation, both of which are pathological features of AD [57]. APP is a transmembrane protein that could be cleaved into Aβ through proteases such as β-secretase and γ-secretase [58]. BACE1 is the functional protein of β-secretase, and NCSTN is one of the core subunits of γ-secretase, which plays a vital role in regulating Aβ generation and may be an important target in AD therapeutics [59]. BACE1 hydrolyzes APP to produce CTF-β, which is then endoproteolyzed by γ-secretase to produce Aβ [60]. The levels of Aβ1-42 and p-Tau/Tau were to be considerably higher in T2DM mice in this investigation. Further study indicated that the mRNA and protein expressions of APP, BACE1 and NCSTN were significantly increased in T2DM mice. In the meantime, we discovered that Rg1 treatment can dramatically downregulate the mRNA and protein expressions of APP, Aβ1-42, BACE1, NCSTN, and p-Tau in the brain of T2DM mice, suggesting that Rg1 may improve DACD by reducing Aβ generation. The results indicated that Rg1 may ameliorate Aβ generation by regulating PLC-TRPC6-mediated Ca2+ homeostasis disorder and further inhibiting CN-NFAT1 signaling in T2DM mice. Furthermore, we found that both Rg1 and metformin therapy could decrease the levels of fasting blood glucose (FBG) as compared with model group, while the FBG was still at a relatively high level including the metformin group. For the hypoglycemic effect, metformin (a positive drug) was significantly better than Rg1, but Rg1 showed a more powerful effect in improving cognitive dysfunction than metformin. We further found that Rg1's powerful ability to improve cognitive dysfunction was consistent with its ability to better alleviate the levels of p-PLC, IP3 and DAG, and their induced calcium overload. Therefore, we speculated that Rg1 treatment may improve DACD by regulating the PLC–CN–NFAT1 signaling pathway instead of decreasing blood glucose in T2DM or other mechanisms. In brief, our findings suggested that Rg1 treatment ameliorates cognitive dysfunction and neuronal lesions, and reduces Aβ generation in the brain of T2DM mice. The process may be involved in the alleviation of NOX2-mediated ROS accumulation, PLC-TRPC6-mediated calcium homeostasis imbalance and CN-NFAT1 activation, which further suppresses APP, BACE1 and NCSTN expression in T2DM mice. Although Rg1 has a dramatic therapeutic efficacy on DACD, more research is still needed to fully recognize the detailed process of Rg1 in the therapy of DACD. All in all, even with limitations, our research supplies a new therapeutic target and potential drug for the treatment of DACD.

The authors declare that no conflicts of interest exist.