Huangdi Anxiao Attenuated High Glucose-Induced PC12 Cells Neurotoxicity via Inhibiting Apoptosis Pathway of Endoplasmic Reticulum Stress

used to treat and its for and a remarkable curative effect. However, the improvement effect of HDAX in the diabetic cognitive dysfunction (DCD) model and the related study explore the neuroprotective effects of and in DCD. A DCD cell model was established by high glucose-induced PC12 cells, and the effect of HDAX on the cell viability was examined by MTT. Additionally, the expression of relevant genes and proteins in the apoptosis pathway of endoplasmic reticulum (ER) stress was detected.


Abstract Background
Huangdi Anxiao (HDAX) is mainly used to treat diabetes and its complications for many years and has a remarkable curative effect. However, the improvement effect of HDAX in the diabetic cognitive dysfunction (DCD) model and the related mechanism is not clear. This study was aimed to explore the neuroprotective effects of HDAX and its possible mechanisms in DCD.
Methods A DCD cell model was established by high glucose-induced PC12 cells, and the effect of HDAX on the cell viability was examined by MTT. Additionally, the expression of relevant genes and proteins in the apoptosis pathway of endoplasmic reticulum (ER) stress was detected.

Conclusions
These results suggested that HDAX had neuroprotective effects in the DCD cell model, which may be associated with the inhibition of the apoptosis pathway of ER stress.

Background
Diabetic cognitive dysfunction (DCD) is a neuropathy caused by sustained hyperglycemia, which is one of the serious complications of diabetes. It is mainly manifested as the decrease of learning and memory ability, accompanied by pathological changes in brain structure and neurological function, which seriously affects the quality of life [1]. Studies have shown that people with diabetes are at least twice as likely to have severe impairment of cognitive function, type 2 diabetes mellitus (T2DM) is a risk for Alzheimer's disease (AD) [2,3]. Although a growing number of researchers have studied over the past few decades, the pathogenesis of DCD remains unclear.
Glucose is the main source of energy, which mammalian brain and its essential function for the normal brain. Recent studies have shown that high glucose is the most fundamental cause of DCD, high glucose environment can lead to increased apoptosis of nerve cells, decrease the ability of learning and memory, and eventually lead to the occurrence of DCD [4]. Besides, chronic hyperglycemia can promote the deposition of Amyloid beta-peptide (Aβ), increase the sensitivity of nerve cells, and thus generate Aβ of neurotoxicity to cerebral microvascular endothelial cells [5]. Some experimental studies showed that high glucose-induced brain insulin resistance leads to changes similar to AD [6]. The exact mechanism of the nerve cell damage caused by high glucose is unclear, but studies have shown that glucose toxicity causes metabolic abnormalities in the following pathways: Endoplasmic reticulum (ER) stress, oxidative stress, in ammatory response, insulin resistance, etc. [7][8][9]. The apoptosis pathway of ER stress is hot in recent years.
The apoptosis pathway of ER stress has been increasingly studied in the occurrence and development of various diabetic complications [10]. ER stress response can be triggered by different factors, such as high glucose environment, in ammatory cytokines, the disorder of calcium balance, oxidative damage, etc. [11], through unfolded protein response (UPR) protect and restore cell function. However, when the injury cannot be recovered in time, UPR can further regulate the downstream apoptotic signaling molecules such as C/EBP homologous protein (CHOP), Cysteinyl aspartate speci c proteinase (Caspase), and B-cell lymphoma-2 (Bcl-2) family to induce apoptosis [12]. In recent years, to nd an effective treatment for DCD, researchers have begun to try to alleviate the disease by regulating the apoptosis pathway of ER stress. Therefore, the development of appropriate drugs has become a hot spot and basic research in a clinic of DCD.
Huangdi Anxiao (HDAX) is a hospital formula from Anhui Provincial Hospital of Chinese Medicine. It is mainly used to treat diabetes and its complications for many years and has a remarkable curative effect [13,14]. However, the improvement effect of HDAX in the DCD model and the related mechanism is not clear. We speculate that HDAX may be effective in the treatment of DCD by inhibiting the apoptosis pathway of ER stress. To elucidate the mechanism of HDAX therapy of DCD, we used high glucose to build DCD vitro model in PC12 cells, to explore the ability of HDAX in neuroprotective effects and related proteins and gens.

Preparation of HDAX containing serum
Twenty rats were divided into two groups randomly: Control group, HDAX group, 10 rats in one group.
Referring to the method of the previous experiment (Cai et al., 2018), HDAX was prepared into the solution at a dose of 10.5 g/kg (7 times of the clinical equivalent dose), the drug was given by gavage of 10 mL/kg for 5 d. The control group was given an equal volume of normal saline. After the last administration, each group was collected blood samples under the abdominal aorta under aseptic conditions, centrifugation for 15 min at 3000 r/min, serum samples were collected, inactivated for 56℃ for 30 min, and stored at -20℃ for later use.
Cell culture and treatments PC12 cells were cultured in Dulbecco modi ed Eagle medium (DMEM, Thermo Fisher Scienti c, Inc., Waltham, USA) in an incubator containing 5% CO 2 at 37 ℃. They were digested by 0.25% pancreatin for passage when the cells reached the con uence rate of above 80%.

Cell viability
Add MTT solution (20 µL, 5 mg/mL) to each hole and incubated, then solubilize the precipitated dye with DMSO. After dissolved for 10-15 min, measured the absorbance at 490 nm with a microplate reader (318C+, Shanghai, China).

The observation of inverted microscope
The cells were seeded onto a six-well plate at a density of 1×10 6 cells/m. The cells were treated with glucose (50, 75, 100, 125, 150 mM) for 48 h. When the cell growing time and density meet the requirement, we selected the clear eld of views to observe the morphology and structure of the cells by inverted microscope.
Hoechst 33342 staining PC12 cells in the logarithmic growth stage were treated according to different groups. After xation with 4% paraformaldehyde for 10 min, 1 mL Hoechst 33342 dye diluent was added to each hole and was

Statistical analysis
The experiment data were expressed as the means±standard deviation (S.D.) (x±s) and analyzed statistically using SPSS 24.0 software (SPSS, Chicago, IL, USA). The differences in the different groups were detected by the one-way ANOVA analysis, which is considered statistically signi cant when P-value<0.05.
Glucose concentration screening PC12 cells were cultured in different densities of glucose (50, 75, 100, 125, 150 mM) for 24, 48, and 72 h, the mannitol balances osmotic pressure between different groups. As shown in Figure 3, Compared with the Control group, when the concentration of building for 100 mM and 125 mM, 48 h, the cells survival rate is close to 50%. But when the mannitol concentration was 125 mM, the cell survival rate was affected (P<0.05). Therefore, according to the MTT results and combined with relevant references, the glucose concentration of 100 mM for 48 h was selected as the suitable experimental condition for the establishment of the DCD cell model in PC12 cells for the subsequent experiment.

Effects of glucose on cell morphology
As shown in Figure 4, After 48 h of culture with different concentrations of glucose, cells in the Control group showed regular morphology, fusiform or polygon, interwoven into a network, adherent to the wall, and good refractive index. With the increase of glucose concentration, the high-glucose injury group was signi cantly damaged in cell morphology, with different degrees of shrinkage, even shedding, and the intercellular space was increased.

Effects of glucose on cell apoptosis
To further determine whether high glucose-induced PC12 cell injury and promoted apoptosis, Hoechst 33342 uorescence staining was used to evaluate the apoptosis of PC12 cells. As shown in Figure 5, after cultured for 48 h with diverse concentrations of glucose, the cells in Control group showed diffuse and uniform low-density uorescence. With the increase of glucose concentration, some of the cells in the high-glucose injury group showed high-density uorescence, and the nucleus appeared to be condensed and fragmented.

HDAX concentration screening
The experiment was divided into seven groups: Control group, different concentrations of HDAX containing serum groups (5%, 10%, 15%, 20%, 25%, 30%), each group respectively incubated 24 hours. As shown in Figure 6, compared with Control group, cell viability had no signi cant difference when the concentration of HDAX containing serum was 5%-15% (P>0.05), When the concentration of HDAX containing serum was 20%, the cell viability began to decline; when the concentration was 30%, the cell viability decreased signi cantly (P<0.05), so 5%, 10%, and 15% dose groups of HDAX containing serum were selected for the next experiment. After treated with high glucose, the cell viability of the 5%, 10% and 15% dose groups of HDAX containing serum was signi cantly higher (P<0.05, P<0.01), among which the 15% dose group of HDAX containing serum had the best effect.
Effects of HDAX on relevant mRNA levels in apoptosis pathway of ER stress on DCD cell model

Discussion
Diabetic cognitive dysfunction (DCD) is a severe diabetes-related complication in the central nervous system [16]. Recent studies have shown that high glucose aggravates neurochemical and structural abnormalities in the brain, which is the most fundamental cause of DCD. Even in non-diabetic patients, impaired glucose tolerance is also an important risk factor for cognitive dysfunction [17]. High glucose can induce neuronal apoptosis and cognitive dysfunction by activating microglial in ammatory response, contributing to the occurrence and development of diabetic encephalopathy and neurodegenerative diseases [18]. It can also change the permeability of blood-brain barrier, thus accelerating the occurrence of cognitive impairment in diabetic patients [1]. As the pathological basis of DCD, it is the research direction of this experiment. In addition, previous studies have shown that HDAX is effective in the treatment of the rat model of DCD [19]. In this experiment, we further study the neuroprotective mechanism of HDAX in the DCD cell model.
First, we took PC12 cells as the main research object, which simulated high glucose and induced neurotoxicity. MTT assay, inverted microscope, and Hoechst 33342 uorescence staining showed that cell viability signi cantly reduced with 100 mM high glucose treatment for 48 h, changed cell morphology, and promoted cell apoptosis. These results indicated that high glucose 100 mM induction for 48 h was the suitable experimental condition for PC12 cells to establish a high glucose injury model in vitro, which was consistent with the research results of other scholars [20,21].
Then, we explored the therapeutic effect of HDAX in DCD. HDAX is rooted in the classical prescription "Xiao-Ke Formula", which is mainly composed of Coptidis Rhizoma, Radix Rehmanniae, Radix Puerariae, Radix Ophiopogonis, Eriobotryae Folium, and Radix Notoginseng. It has been used in the treatment of diabetes and its complications for several years, but it has rarely been studied in the research on DCD. We reanalyzed the HDAX pharmacological formula from the perspective of modern pharmacological research. In this prescription, Berberine, the main component of Coptidis Rhizoma, can ameliorate rats model of combined Alzheimer's disease and type 2 diabetes mellitus via the suppression of ER stress [22]. Studies in Radix Ophiopogonis showed that it reducing cell apoptosis, protecting islets, and improving insulin resistance [23]. Panax notoginseng saponins are the major active ingredients of Radix Notoginseng, which can keep neurons from oxidative stress damage, ameliorate learning and memory de cits in AD rats [24]. Therefore, we proposed a hypothesis that HDAX can improve DCD. We studied the neuroprotective effect of HDAX on DCD for the rst time in vitro. In this experiment, we proved that HDAX can improve the cell vitality of the DCD cell model and exert its neuroprotective effect.
Next, we further explored whether the mechanism of HDAX protecting neurons is related to the inhibition of the apoptosis pathway of ER stress. We selected the related proteins and genes in this pathway for comparison, and the results were consistent with our expectations. Glucose regulated protein 78 (GRP78) was a symbolic protein and protective factor of ER stress and had the function of protecting the endoplasmic reticulum under stress [25]. CHOP was extensively induced and expressed under ER stress, and nally induces apoptosis through the mitochondrial pathway [26]. Bcl-2-associated X protein (Bax) was a pro-apoptotic protein that promoted the occurrence of apoptosis, and Bcl-2 was an anti-apoptotic protein, inhibited the occurrence of apoptosis. They usually existed in the form of heterodimers and jointly regulated the process of cell apoptosis [27]. Caspases were the core component of the apoptosis reaction. Caspase-12 was a speci c apoptosis mediator in ER stress-mediated apoptosis pathway. Under normal circumstances, Caspase-12 bonded to ER membrane or formed a hetero with tumor necrosis factor receptor-associated factor 2 inactive. However, in the case of the excessive ER stress response, Caspase-12 activates downstream molecules Caspase-9 and Caspase-3 [28]. Caspase-3 was an important protease for the executive function of apoptosis. It was located downstream of the Caspase cascade and was considered to be the endpoint of apoptosis [29]. In this experiment, Our ndings showed that HDAX can improve the relevant protein expression and mRNA levels after treatment. Moreover, treatment with 4-phenylbutyric acid (4-PBA), as well as cotreatment with HDAX and 4-PBA, signi cant inhibition of cell damage, which further demonstrated that the potential mechanism of HDAX to improve DCD by restraining the apoptosis pathway of ER stress.

Conclusions
Our ndings suggest that HDAX was con rmed with a protective effect on DCD models, which might be associated with the inhibition of the apoptosis pathway of ER stress. However, there still needs deeper exploration. These data may help to explain the potential neuroprotective mechanism of HDAX on DCD.