Role of IGF1/IGFR signaling pathway in neuroprotective effect of Shengmai Dihuang Decoction on CIH-induced cognitive impairment

Abstract

Background

As the most important pathophysiological process of obstructive sleep apnea (OSA), chronic intermittent hypoxia (CIH) could cause cognitive impairment. Our previous study showed that Shengmai Dihuang Decoction (SDD) improved CIH-induced cognitive impairment. However, the precise underlying mechanism remains unclear.

Objective

This study aims to investigate the role of the insulin-like growth factor 1 (IGF1)/insulin-like growth factor 1 receptor (IGFR) signaling pathway in improving CIH-induced neuronal injury by SDD treatment.

Methods

In this study, HT22 cells were cultured under CIH conditions (O₂: 21–0.1 %, 6 cycles/h). Cell ability was assessed by CCK-8 assay, and Mitochondrial damage was detected by transmission electron microscopy (TEM), mitochondrial membrane potential (MMP), ATP, and mitochondrial complex I activity assay. Mitochondrial oxidative stress was detected by MitoSOX superoxide indicator. IGF1/IGFR signaling pathways were detected by Elisa, immunofluorescence staining, and Western blot. In addition, C57BL/6 mice were exposed to CIH for 35 days (O₂: 21-5%, 8 h/d), and cognitive impairment was assessed by Morris water maze.

Results

Results showed that cell activity was decreased during CIH exposure, followed by mitochondrial damage and decreased IGF1/IGFR expressions. Exogenous IGF1 treatment increased cell activity and improved mitochondrial dysfunction. SDD treatment increased IGF1/IGFR expressions, activated the RAS/RAF/MAPK and PI3K/AKT signaling pathways, increased ERK/CREB/BDNF and postsynaptic density protein 95 (PSD-95) expressions, and improved mitochondrial damage and cell activity. IGF1 siRNA or AG1024 (IGFR antagonist) reversed the protective effects of SDD under CIH condition.

Conclusion

Results suggest that SDD alleviates CIH-induced neuronal injury, mainly by improving mitochondrial damage via activation of IGF1/IGFR signaling pathway.

Graphical abstract

1. Introduction

The respiratory condition known as obstructive sleep apnea (OSA) is characterized by recurring bouts of upper airway collapse that result in chronic intermittent hypoxia while asleep.¹ Researchers have demonstrated a strong correlation between OSA and cognitive decline,² and as the most important pathophysiological process of OSA, leads to neuronal damage.³ CIH accelerates tau protein dispersion and phosphorylation, exacerbating memory deficits and impairments in synaptic plasticity.⁴ CIH can lead to neuronal apoptosis and synaptic plasticity injury in the cortex and hippocampus.⁵ Synaptic plasticity is the basis for changes in learning and memory, which in turn cause an increase in the number and volume of synapses and the formation of new neural circuits.⁶ Additionally, synaptic plasticity and neuronal survival are both significantly influenced by mitochondrial function. CIH exposure results in mitochondrial dysfunction, decreased ATP synthesis, and increased mitochondrial depolarization and reactive oxygen species (ROS) release.³ In recent years, evidence has shown that increased oxidative stress is related to neurocognitive impairment in OSA.⁷ Oxidative stress is caused by elevated intracellular levels of ROS, which can lead to mitochondrial damage and ultimately neuronal damage.⁸ However, the mechanism of CIH-induced neuronal damage is not fully understood.

Insulin-growth factor 1 (IGF1) belongs to the family of insulin-like peptides (ILPs) and is an important growth factor for the central nervous system.⁹ The main functions of IGF1 in the brain are related to cell growth, differentiation, maturation, metabolic processes, and synaptic plasticity.¹⁰ IGF1 deficiency leads to impaired neuronal excitability and changes in synapse-associated proteins such as brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95), cyclic AMP-response element binding protein (CREB), and extracellular signal-related kinase (ERK).¹¹ Administration of IGF1 improves glutamatergic synaptic transmission efficacy in hippocampal circuits and enhances learning and memory.¹⁰ When IGF1 binds with insulin-growth factor 1 receptor (IGFR), receptor dimerization occurs, which in turn activates downstream signaling pathways such as phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and RAS/RAF/mitogen -activated protein kinase. IGFR initiates the downstream IGF1 signaling pathway, enhances the transmission and integration of glutamate receptors to dendritic spines, and maintains synaptic function.¹²

In traditional Chinese medicine (TCM), cognitive impairment belongs to the category of “dementia.”⁵ In TCM, the kidney has the functions of storing essence, main growth and development, and producing marrow.¹³ The marrow is connected with the brain. The brain is located in the cranial cavity and is formed by the convergence of the marrow. Kidney essence can replenish the brain and promote brain development.¹⁴ Kidney deficiency is a noun in TCM, which refers to the deficiency of kidney essence, Qi, Yin and Yang.¹⁵ Studies have found that kidney deficiency affects brain morphology,¹⁶ brain function,¹⁷ leading to hippocampal neurotransmitter transcription disorders and neuronal apoptosis, thereby inducing a variety of neurological diseases.¹⁸ The kidney-tonifying method has been shown to improve cognitive dysfunction by increasing hematopoietic growth factors.¹⁹

In TCM, Qi is a very subtle substance that constitutes the human body. Qi is divided into Yin and Yang. Yin and Yang indicate two opposite specific attributes, such as light and dark, surface and inside, cold and heat and so on. The interaction of Yang Qi and Yin Qi promotes human metabolism.²⁰ Kidney deficiency leads to cognitive dysfunction, often with qi and yin deficiency symptoms. Deficiency of both Qi and Yin is a common pathological state in TCM, which describes the coexistence of Qi deficiency and Yin deficiency in human body. And shows symptoms such as thirst, weight loss and irritability.²¹

Shengmai Dihuang Decoction (SDD) was originally used to treat the lung and kidney Qi deficiency type of “consumptive thirst” in ancient Chinese medicine, which is referred to as type 2 diabetes mellitus in modern medicine. “Consumptive thirst” has clinical manifestations of lung and kidney Qi and Yin deficiency and bone marrow deficiency. According to Chinese medicine, kidney deficiency causes mental deficiency, leading to impaired cognitive ability.²² SDD is effective for the treatment of lung and kidney Qi deficiency in the form of “consumptive thirst,” and has been shown to alleviate renal impairment in CIH-exposed mice.^21,23 SDD is composed of two kinds of Chinese medicine, Shengmai San and Liuwei Dihuang Decoction. Liuwei Dihuang Decoction is a classic formula for nourishing Yin and tonifying the liver and kidneys. Studies have shown that Liuwei Dihuang Decoction can improve cognitive impairment, promote neurogenesis, alleviate neuronal apoptosis, and facilitate synaptic plasticity in a mouse model of aging by regulating the IGF1 signaling pathway.24, 25, 26 Shengmai San has the effects of replenishing qi and restoring pulse, nourishing yin, and generating fluid. Emaciation, weakness, anorexia, thirst, and irritability were all symptoms of Qi and Yin deficiency in our prior study of mice exposed to CIH.^5,27 The study also showed that the main components in SDD, catalpol and ginsenosides, have significant effects on alleviating neuronal damage, maintaining the transmission frequency of neurotransmitters, enhancing neuronal synaptic transmission efficiency, and ameliorating cognitive dysfunction.28, 29, 30

First of all, the purpose of this study was to determine whether SDD has a protective effect on neuronal damage induced by CIH exposure. Secondly, the aim was to explore the role of IGF1/IGFR signaling pathway in neuroprotection.

2. Materials and methods

2.1. Cell culture

HT22 cells were randomly assigned to the following groups: control (CON), chronic intermittent hypoxia (CIH), and low, medium, and high SDD treatment groups (SDD-L, SDD-M, SDD-H). The CON group was exposed to normoxic conditions, and the other groups were subjected to CIH (O₂ concentration in the hypoxic chamber ranged from 1 % to 21 %, 10 min/cycle).

2.2. Reagents and antibodies

Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), and Dulbecco's Modified Eagle's Medium were purchased from Gibco Invitrogen Corporation (Carlsbad, CA, USA). RIPA lysis buffer and penicillin streptomycin solution were obtained from Solibao Technology Co., Ltd. (Beijing, China). IGF1 (bs-0014R), IGFR (bs-4985R), P-CREB (bs-5256R), CREB (bsm-33196R), P-ERK (bs-3016R), and ERK (bsm-52259R) were purchased from Bioss Antibodies (Woburn, MA, USA). RAS (DF6324), RAF (AF0837), and MAPK (AF6456) were purchased from Affinity (West Bedford, UK). MAP2 (67015-1-lg), PI3K (67071-1-lg), P-AKT (66444-1-lg), and AKT (60203-1-lg) were bought from Proteintech (Rosemont, IL, USA). GSK3β (GB11099), PSD-95 (GB11277), BDNF (GB11559), β-Actin (GB12001), and β-Tubulin (GB12139) were purchased from Servicebio (Wuhan, China).

2.3. Preparation of SDD

The composition of SDD was shown in Table 1. The above plant names can be found at www.worldfloraonline.org. SDD was purchased from ShenWei Pharmaceutical Group Co., Ltd. (Shijiazhuang, China). The SDD crude was steeped in ultrapure water for 0.5 h before boiling twice for 1 h each and finally being concentrated to 100 mL.

Table 1.

Prescription of Shengmai Dihuang Decoction.

Local Name	English name	Latin name	Part used	Origin (P.R.China)	quantity (g)
Di Huang	Radix Rehmanniae	Rehmannia glutinosa (Gaertn.) DC.	Root	He Bei	24g
Ren Shen	Ginseng	Panax ginseng C. A. Mey.	Root	Ji Lin	9g
Maimendong	Ophiopogonis	Ophiopogon japonicus (Thunb.) Ker Gawl.	Root	Zhe Jiang	9g
Wu Wei Zi	Schisandrae	Schisandra chinensis (Turcz.) Baill.	Seed	Hei Long Jiang	6g
Mu Danpi	Cortex Moutan	Paeonia suffruticosa Andrews	Root	An Hui	9g
Shan Zhuyu	Fructus Corni	Cornus officinalis Siebold & Zucc.	Pulp	Zhe Jiang	12g
Fu Ling	Poria Cocos	Poria cocos (Schw.) Wolf.	sclerotium	An Hui	9g
Ze Xie	Rhizoma Alismatis	Alisma orientalis (Sam.) Juzep.	stem tuber	Fu Jian	9g
Shan Yao	Rhizoma Dioscoreae	Dioscorea opposita Thunb.	Root	He Nan	12 g

The components in water extract were qualitatively analyzed by HPLC (Fig. 1C–D). Using the gradient elute technique, acetonitrile (A) and 0.1 % acetic acid (B) were used to create the mobile phase (0–10 min, A: 5 %. 10–25 min, A: 15 %–25 %. 25–35 min, A: 25 %–45 %. 35–45 min, A: 45 %–55 %, 45–55 min, A: 55 %–60 %. 55–60 min, A: 60 %–60 %). The column size was 250 ∗ 4.6 mm. Analysis column using 5 μm filler. Wavelength, 210 nm. Sample volume, 10 μL. Catalpol, morroniside, loganin, ginsenoside Rb1, and paeonol were purchased from Shanghai Yuanye Biological Co., Ltd.

Fig. 1.

(A) Mechanism of SDD alleviating neuronal injury in CIH-exposed HT22 cells by IGF1/IGFR signaling pathway. (B) Role of IGF1/IGFR signaling pathway in alleviating CIH induced cognitive impairment by SDD treatment. (C) Mixed drug standards. (D) SDD water extract. 1: Catalpol, 2: Morroniside, 3: Loganin, 4: Ginsenoside Rb1, 5: Paeonol. Created with BioRender. com.

2.4. Preparation of SDD-containing serum

Gavage groups of 30 male C57BL/6 mice were distributed evenly and randomly among the SDD treatment groups. The clinical dose of SDD is 99 g/day. The weight of adults is about 60 kg. Based on body surface area, the conversion coefficient (Km) for converting human dosages to mice is 12.3.³¹ The mice equivalent dose: 99 g/60 kg × 12.3 = 20 g/kg. Thus, the medium dose of SDD was 20 g/kg/day. Therefore, in this study, the low dose was 10 g/kg/day, and the high dose was 30 g/kg/day.

Mice were intragastrically administered SDD (10, 20, 30 g/kg) for 7 days. At 1 h after the end of gavage on day 7, blood was taken from the inner canthus of the mice, and the serum containing SDD was obtained by centrifugation, inactivation, and sterilization.

2.5. CCK-8 assay

Briefly, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and cultured for 48 h. Then 10 μL CCK-8 reagent was added to each well and cells were cultured for another 30 min. The absorbance is 450 nm.

2.6. Electron microscopy

The HT22 cells were digested with 0.25 % trypsin, centrifuged, and resuspended in fixative solution. After dehydration and embedding, 1–2 μm semi-thin sections were made, and then 60–80 nm ultrathin sections were made followed by double staining with uranyl acetate and lead nitrate.

2.7. Western blot analysis

HT22 cells were lysed. The supernatant was collected after centrifugation (12000 g, 20 min, 4 °C), and the total protein content was measured using a BCA kit. Then protein was electrophoretically resolved and wet transferred to a PVDF membrane. The membrane was incubated at 4 °C overnight with primary antibodies against IGF1, IGFR, BDNF, PSD-95, P-ERK, ERK, P-CREB, CREB, RAF, RAS, MAPK, PI3K, P-AKT, AKT, GSK3β, β-Actin and β-Tubulin. The membrane strips were washed wtih TBST. Then the strips were incubated with horseradish peroxidase-linked secondary antibody. Proteins were detected by enhanced chemiluminescence and quantitated with ImageJ software.

2.8. Immunofluorescence staining

After HT22 cells reached the logarithmic growth phase, they were dissociated from the Petri dish with 0.25 % trypsin and centrifuged, after which the supernatant was discarded. The cells were seeded in 12-well plates. After 48 h, the cells were fixed in 4 % paraformaldehyde for 15 min. The fixed cells were blocked in anti-goat serum at 37 °C for 1 h. Then the serum was removed, and cells were incubated with primary antibodies against MAP2 and IGFR at 4 °C. Next, the cells were rinsed with PBS, followed by incubation with fluorescence-coupled secondary antibody for 1 h at 37 °C in the dark. Then the cells were washed with PBS. Finally, the sealant containing DAPI was added dropwise.

2.9. Elisa

To perform the enzyme-linked immunosorbent assay (ELISA), HT22 cells were collected and centrifuged. Then the concentration of IGF1 in the culture medium of HT22 cells was detected using the Mouse IGF-1 ELISA Kit according to the manufacturer's instructions.

2.10. IGF1 small interfering RNA

HT22 cells were seeded in 6-well plates. The transfection complexes were prepared according to the manufacturer's instructions (RiboBio Co., Guangdong, China). Then the transfection complexes were added to the culture medium. The IGF1 small interfering RNA (siRNA) sequence was (F): 5-GCAAUUUACUCAUUGUUUA-3′; and (R): 5′-UAAACAAUGAGUAAAUUGC-3′.

2.11. Mitochondrial ROS

According to the instructions (M36008, Invitrogen™), briefly, the cells were seeded at a density of 1 × 10⁴ cells/well in 12-well plates. Then the cells were incubated with the MitoSOX Red probe for 30 min in the dark. Finally, the cells were washed with Hank's Balanced Salt Solution.

2.12. Mitochondrial membrane potential (MMP)

According to the instructions (G1515-100T, Servicebio), HT22 cells were seeded in 12-well dark plates at a density of 1 × 10⁵ cells/well. After being stained with JC-1 and allowed to sit in the working solution for 30 min, cells were examined by fluorescence microscopy.

2.13. Measurement of ATP

Using the lysis buffer included in the kit (Beyotime, Jiangsu, China), cells were collected and lysed. After centrifugation at 12000g, 4 °C. ATP detection reagent was added to the supernatant. After 5 min, several samples and standards were placed in a 96-well plate. The RUL was measured with a multimode microplate reader.

2.14. Mitochondrial respiratory chain complex I

The activities of respiratory chain enzyme complex I in mitochondria were assayed using a respiratory chain complex assay kit (Abbkine, Wuhan, China). HT22 cells were collected and mitochondria were extracted. Then the relevant reagents were added to the lysed mitochondria. Finally, the absorbance was read at 340 nm.

2.15. Animals

Adult male C57BL/6 mice (6–8 weeks, 20–22 g) were randomly divided into 5 groups: CON group, CIH group, SDD group, SDD + siRNA group and SDD + AG1024 group. The mice in the CON group were placed in a normoxic environment, and the other four groups of mice were placed in a hypoxic chamber. The gas control system was connected to the chamber to reduce the oxygen concentration in the chamber from 21 % to 5 % and then from 5 % to 21 %, 8 h/d (9:00–17:00) for 35 days. Mice in the SDD group, SDD + siRNA group and SDD + AG1024 group were given SDD (30 g/kg) by gavage 30 min before entering the hypoxic chamber every day (approved by Hebei University of Chinese Medicine Animal Care and Use Committee, No. DWLL2020044). At the same time, IGF1 siRNA or AG1024 were injected into the intracerebroventricular of mice. The CIH animal model was prepared with reference to the relevant literature.¹⁹

2.16. Intracerebroventricular injection

After anesthesia, the mice were fixed on the brain stereotaxic apparatus and IGF1 siRNA (0.2 nM, 2 μL) or AG1024 (10 mM, 2 μL) were injected into the lateral ventricle (anteropos-terior - 0.34 mm, lateral −1.0 mm, and depth −2.2 mm). Following the experiment, the accuracy of the injection point was verified using 2 % Chicago Blue 6B (2 μL).

2.17. Morris water maze (MWM)

MWM consisted of a circular pool, a platform under the water surface, and an image acquisition system. The pool space was divided into four virtual quadrants. The escape latency, swimming speed, the number of crossing the platform, the distance and time in the target quadrant of the mice from the starting position to the platform were recorded. The real-time tracking software was used to record the behavioral trajectories of mice above the pool after removing the platform. The specific experimental methods refer to our previous research.⁵

2.18. Statistical analyses

Data were analyzed by SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA), and are presented as the mean ± standard deviation. Using ANOVA, P < 0.05 was considered statistically significant.

3. Results

3.1. Qualitative analysis of SDD

Components of SDD were qualitatively analyzed by HPLC. Mixed standard drug as: Catalpol, Morroniside, Loganin, Ginsenoside Rb1, Paeonol were shown in Fig. 1C. As seen in Fig. 1D, Catalpol, Morroniside, Loganin, Ginsenoside Rb1, and Paeonol were found to be significantly present in the water extract of SDD.

3.2. CIH exposure inhibited the activity of HT22 cells and decreased IGF1 and IGFR expression

CIH exposure could lead to hippocampal injury associated with cognitive impairment. First, authors evaluated the activity of HT22 cells during CIH exposure (0–48 h). As shown in Fig. 2A, cell activity was decreased after 12, 24, and 48 h of CIH stimulation (P < 0.001). IGF1 in the brain is related to cell growth, differentiation, maturation, metabolic processes, and synaptic plasticity. Thus, IGF1/IGFR expression was observed under CIH conditions. According to Fig. 2B, IGF1 expression in HT22 cells was decreased after 48 h of CIH stimulation (P < 0.05). The results indicated in Fig. 1C that the level of IGF1 in the cell culture media was markedly decreased after 24 and 48 h of CIH stimulation (P < 0.05 or P < 0.01). As shown in Fig. 2D and E, the positive cells of IGFR in HT22 were significantly decreased after 24 and 48 h of CIH stimulation (P < 0.05 or P < 0.01). As shown in Fig. 2F, IGFR protein expression in HT22 cells was significantly decreased after 48 h of CIH stimulation (P < 0.01). Thus, CIH exposure for 48 h was chosen for subsequent experiments.

Fig. 2.

Effects of CIH exposure on cell activity and IGF1/IGFR expression in HT22 cells. (A) Effects of CIH exposure on cell viability at different times. (B) IGF1 expression in HT22 cells at various CIH exposure times. (C) Secretion levels of IGF1 in cell cultures at different CIH exposure times. (D–E) HT22 cells expressing IGFR were detected by immunofluorescence at different CIH exposure times; arrows denote the positive cells (scale bar = 25 μm). (F) IGFR expression in HT22 cells after various CIH exposure times. The outcomes are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus 0 h CIH exposure.

3.3. Exogenous IGF1 improved cell viability and mitochondrial dysfunction in CIH-exposed HT22 cells

Exogenous IGF1 treatment played a crucial role in alleviating cell damage and mitochondrial dysfunction.³² Studies have found that aging rats without IGF1 treatment showed significant mitochondrial dysfunction, including decreased ATPase and respiratory chain complex activity.³³ This suggests that IGF1 could be a critical target for alleviating neuronal injury and mitochondrial dysfunction induced by CIH exposure. Therefore, our study assessed whether exogenous IGF1 is beneficial for improving neuronal activity and mitochondrial damage under CIH conditions. As shown in Fig. 3A, the activity of HT22 cells was decreased in the CIH group (P < 0.001 versus the CON group), whereas after IGF1 treatment at 0.1, 0.3, and 1 μg/mL, the activity of HT22 cells was clearly increased (P < 0.01 or P < 0.001 versus the CIH group). Authors chose 0.1 μg/mL of IGF1 for subsequent experiments. Mitochondrial metabolism disorder and oxidative stress may be the main causes of neuronal damage under CIH conditions.³⁴ Thus, authors assessed the effect of IGF1 on mitochondrial dysfunction by transmission electron microscopy (TEM), as well as the JC-1 mitochondrial membrane potential assay, ATP assay, CheKine™ Micro Mitochondrial complex I activity assay, and the MitoSOX™ Red superoxide indicator under CIH conditions. As shown in Fig. 3B, exogenous IGF1 treatment significantly improved mitochondrial damage under CIH conditions. Fig. 3C–H showed that MMP, ATP level, and mitochondrial respiratory chain complex Ι level were significantly decreased in CIH-exposed HT22 cells (P < 0.01 or P < 0.001 versus the CON group), but ROS levels in mitochondria were increased in CIH-exposed HT22 cells (P < 0.001 versus the CON group). Treatment with 0.1 μg/mL IGF1 alleviated mitochondrial dysfunction (P < 0.05 or P < 0.01 versus the CIH group). Fig. 3I–L indicated that the expression of BDNF, PSD-95, P-CREB, and P-ERK was markedly decreased in the CIH group (P < 0.05, P < 0.01 or P < 0.001 versus the CON group); whereas treatment with 0.1 μg/mL IGF1 increased protein expression (P < 0.05 or P < 0.001 versus the CIH group).

Fig. 3.

Effects of exogenous IGF1 on cell viability and mitochondrial dysfunction in CIH-exposed HT22 cells. (A) Effects of various IGF1 concentrations on cell viability. (B) TEM was used to detect mitochondria in HT22 cells that had been exposed to CIH (20000 × ). (C and E) Effects of exogenous IGF1 on the mitochondrial membrane potential of HT22 cells (scale bar = 50 μm). (D and F) Effects of exogenous IGF1 on ROS production in the mitochondria of CIH-exposed HT22 cells (scale bar = 25 μm). (G) Effects of exogenous IGF1 on mitochondrial ATP levels in CIH-exposed HT22 cells. (H) Effects of exogenous IGF1 on the level of mitochondrial respiratory chain complex Ι in HT22 cells exposed to CIH. (I) BDNF expression in CIH-exposed HT22 cells. (J) PSD-95 expression in CIH-exposed HT22 cells. (K) P-CREB/CREB expression in HT22 cells exposed to CIH. (L) P-ERK/ERK expression in CIH-exposed HT22 cells. The results are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the CON group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the CIH group.

3.4. SDD increased cell viability and improved mitochondrial dysfunction in CIH-exposed HT22 cells

A previous study showed that Liuwei Dihuang Decoction exerts tonifying kidney effects by regulating IGF1.²⁶ Shengmai San has significant anti-Qi and Yin deficiency effects, in line with the basic pathogenesis of CIH exposure.^35,36 Therefore, under CIH circumstances, the effects of SDD on cell survival and mitochondrial dysfunction were evaluated. According to TEM findings, as shown in Fig. 4A, treatment with SDD-containing serum improved the impairment of mitochondria in CIH-exposed HT22 cells. Fig. 4B to G showed that the MMP, ATP level, and mitochondrial respiratory chain complex Ι level in HT22 cells were decreased after CIH exposure (P < 0.01 or P < 0.001 versus the CON group), and ROS level was increased in CIH-induced HT22 cells (P < 0.05 versus the CON group), all of which were reversed after treatment with medium or high doses of SDD-containing serum (P < 0.05, P < 0.01 or P < 0.001 versus the CIH group). Fig. 4H showed that the HT22 cells activity was markedly decreased after CIH exposure (P < 0.001 versus the CON group), which was improved after treatment with high doses of SDD-containing serum (P < 0.001 versus the CIH group). Fig. 4I–L showed that the expression of PSD-95, BDNF, P-CREB and P-ERK was significantly decreased after CIH exposure (P < 0.05 versus the CON group), whereas the protein expression was increased after treatment with medium or high doses of SDD-containing serum (P < 0.05, P < 0.01 or P < 0.001 versus the CIH group).

Fig. 4.

Effects of SDD on cell viability and mitochondrial dysfunction under CIH circumstances in HT22 cells. (A) TEM was used to detect mitochondria in CIH-exposed HT22 cells (20000 × ). (B and D) Impact of SDD on the potential of mitochondria in CIH-exposed HT22 cells (scale bar = 50 μm). (C and E) Effects of SDD on mitochondrial ROS in CIH-exposed HT22 cells (scale bar = 25 μm). (F) Effects of SDD on mitochondrial ATP levels in CIH-exposed HT22 cells. (G) Effects of SDD on the level of mitochondrial respiratory chain complex Ι in CIH-exposed HT22 cells. (H) Cell viability after CIH exposure. (I–J) PSD-95 and BDNF expression in CIH-exposed HT22 cells. (K–L) P-CREB/CREB and P-ERK/ERK expression in CIH-exposed HT22 cells. The results are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the CON group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the CIH group.

3.5. SDD increased the expression of IGF1/IGFR downstream signaling molecules under CIH conditions

Authors assessed whether the neuroprotective effects of SDD are related to regulation of the IGF1/IGFR signaling pathway under CIH conditions. Fig. 5A to C showed that the level of IGF1 secretion in cell culture media and IGF1 expression in HT22 cells were decreased after CIH exposure (P < 0.05 or P < 0.001 versus the CON group), but were increased after treatment with medium or high doses of SDD-containing serum (P < 0.05 or P < 0.01 versus the CIH group). According to Fig. 5D–F, the number of HT22 cells expressing IGFR was significantly decreased after CIH exposure (P < 0.01 or P < 0.001 versus the CON group), but was increased after treatment with medium or high doses of SDD-containing serum (P < 0.01 versus the CIH group).

Fig. 5.

Effect of SDD on IGF1 and IGFR expression in HT22 cells under CIH conditions. (A) Western blotting of IGF1. (B) IGF1 expression in CIH-exposed HT22 cells. (C) Secretion levels of IGF1 in cell cultures exposed to CIH. (D–E) The number of IGFR-positive cells was detected by immunofluorescence in CIH-exposed HT22 cells; arrows denote the presence of positive cells (scale bar = 25 μm). (F) IGFR expression in CIH-exposed HT22 cells. The results are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the CON group, ^#P < 0.05, ^##P < 0.01 versus the CIH group.

The PI3K/AKT/GSK3β and RAS/RAF/MAPK signaling pathways are the two major downstream signaling pathways of IGFR, which play important roles in neuronal damage under CIH conditions. Fig. 6A to G showed that the expression of RAS, RAF, MAPK, PI3K, P-AKT in CIH-exposed HT22 cells was clearly decreased (P < 0.05, P < 0.01 or P < 0.001 versus the CON group), but was increased after treatment with medium or high doses of SDD-containing serum (P < 0.05, P < 0.01 or P < 0.001 versus the CIH group). It is worth noting that GSK3β is a direct substrate of PI3K/AKT, and its overexpression affects neurogenesis and aggravates neuronal damage.³⁷ As shown in Fig. 6H, GSK3β expression was markedly increased after CIH stimulation (P < 0.05 versus the CON group), whereas treatment with medium and high doses of SDD-containing serum decreased GSK3β expression (P < 0.05 versus the CIH group).

Fig. 6.

Effects of SDD on the IGF1/IGFR signaling pathway under CIH conditions. (A) Western blotting RAS, RAF, and MAPK. (B–D) RAS, RAF, and MAPK expression in CIH-exposed HT22 cells. (E) Western blotting of PI3K, P-AKT/AKT, and GSK3β in CIH-exposed HT22 cells. (F–H) PI3K, P-AKT/AKT, and GSK3β expression in CIH-exposed HT22 cells. The results are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the CON group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the CIH group.

3.6. IGF1 siRNA and AG1024 inhibited the neuronal protective effects of SDD under CIH conditions

To confirm that SDD improved neuronal damage under CIH conditions by regulating the IGF1/IGFR signaling pathway, IGF1 siRNA and the AG1024 IGFR antagonist were employed. As shown in Fig. 7A to G, the expression of RAS, RAF, MAPK, PI3K, and P-AKT in the CIH group was decreased (P < 0.05, P < 0.01 or P < 0.001 versus the CON group), but was significantly increased after treatment with high doses of SDD-containing serum (P < 0.05, P < 0.01 or P < 0.001 versus the CIH group). As shown in Fig. 7H, CIH exposure increased the expression of GKS3β (P < 0.001 versus the CON group), whereas treatment with high doses of SDD-containing serum decreased its expression (P < 0.01 versus the CIH group). However, pre-treatment with IGF1 siRNA or AG1024 reversed the protein expression caused by high doses of SDD-containing serum (P < 0.05, P < 0.01 or P < 0.001 versus the SDD group).

Fig. 7.

Role of IGF1 siRNA and AG1024 in regulating the effects of SDD on the expression of IGF1/IGFR downstream signaling molecules in CIH-exposed HT22 cells. (A) Western blotting of RAS, RAF, and MAPK. (B–D) Expression of RAS, RAF, and MAPK in CIH-exposed HT22 cells. (E) Western blotting of PI3K, P-AKT/AKT, and GSK3β in CIH-exposed HT22 cells. (F–H) Expression of PI3K, P-AKT/AKT, and GSK3β in CIH-exposed HT22 cells. The results are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the CON group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the CIH group, ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus the SDD group.

As shown in Fig. 8A to D, CIH stimulation significantly decreased the expression of BDNF, PSD-95, P-CREB, and P-ERK. (P < 0.05 or P < 0.01 versus the CON group). An increase in expression of the aforementioned proteins was also observed after treatment with high doses of SDD-containing serum (P < 0.05 or P < 0.01 versus the CIH group). However, pre-treatment with IGF1 siRNA or AG1024 decreased protein expression and reversed the mitochondrial improvement caused by treatment with high doses of SDD-containing serum (P < 0.05 or P < 0.01 versus the SDD group; Fig. 8E). Fig. 8F to K showed that the mitochondrial membrane potential, ATP level, and mitochondrial respiratory chain complex Ι level in HT22 cells were significantly decreased under CIH conditions (P < 0.01 or P < 0.001 versus. the CON group); however, ROS level in CIH-exposed HT22 cells was increased (P < 0.01 versus CON group). Trends of the above indexes were reversed after treatment with high doses of SDD-containing serum (P < 0.05 or P < 0.01 versus. the CIH group), but pre-treatment with IGF1 siRNA or AG1024 reversed these therapeutic effects (P < 0.05, P < 0.01 or P < 0.001 versus the SDD group). The viability of CIH-exposed HT22 cells was significantly decreased (P < 0.001 versus the CON group). However, treatment with high doses of SDD-containing serum caused a significant increase in cell survival (P < 0.05 versus the CIH group), which was blocked by pretreatment with IGF1 siRNA or AG1024 (P < 0.01 or P < 0.001 versus the SDD group; Fig. 8L).

Fig. 8.

Role of IGF1 siRNA and AG1024 in improving the effects of SDD on neuronal injury in CIH-exposed HT22 cells. (A–B) PSD-95 and BDNF expression in CIH-exposed HT22 cells. (C–D) P-CREB/CREB and P-ERK/ERK expression in CIH-exposed HT22 cells. (E) TEM was used to detect mitochondria in CIH-exposed HT22 cells (20000 × ). (F and H) Mitochondrial membrane potential in CIH-exposed HT22 cells (scale bar = 50 μm). (G and I) Mitochondrial ROS in CIH-exposed HT22 cells (scale bar = 25 μm). (J) Mitochondrial ATP levels in CIH-exposed HT22 cells. (K) The level of mitochondrial respiratory chain complex Ι in CIH-exposed HT22 cells. (L) The viability of CIH-exposed HT22 cells. The results are presented as the mean ± standard deviation (SD). n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus the CON group, ^#P < 0.05, ^##P < 0.01 versus the CIH group, ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus the SDD group.

3.7. IGF1 siRNA and AG1024 reversed the improvement of SDD treatment on cognitive deficit in CIH-exposed mice

MWM is one of the methods to evaluate hippocampus-dependent spatial learning and memory in rodents. MWM was used to evaluate the effects of IGF1 siRNA and AG1024 on cognitive impairment in CIH-exposed mice. As shown in Fig. 9A, on days three to five, the mice in the CIH group had a substantially longer escape latency than the mice in the CON group (P < 0.001 versus the CON group). The escape latency was dramatically reduced following SDD therapy (P < 0.01 versus the CIH group). However, intracerebroventricular injection of AG1024 or IGF1 siRNA significantly prolonged the escape latency in comparison with the SDD group (P < 0.05 or P < 0.01 versus the SDD group). As seen in Fig. 9B, the lack of a significant difference in swimming speed across the groups suggested that neither CIH exposure nor lateral ventricle injection affected the capacity of mice to move about. As shown in Fig. 9C–D, CIH exposure in mice significantly reduced the number of crossing the platform (P < 0.001 versus the CON group), but it increased significantly after SDD treatment (P < 0.05 versus the CIH group). However, the number crossing the platform was significantly reduced after intracerebroventricular injection of IGF1 siRNA or AG1024 (P < 0.05 versus the SDD group). As shown in Fig. 9E–F, the distance and time of mice in the target quadrant were significantly decreased after CIH exposure (P < 0.001 versus the CON group), and they were significantly increased after SDD treatment (P < 0.01 or P < 0.001 versus the CIH group). However, IGF1 siRNA or AG1024 treatment reversed the therapeutic effect of SDD (P < 0.01 or P < 0.001 versus the SDD group). Fig. 9G–H showed that in the visible platform test, the escape latency and swimming speed of each group were not statistically significant (see Fig. 10).

Fig. 9.

Role of IGF1 siRNA and AG1024 in the improvement effects of SDD by Morris water maze in CIH-exposed mice. (A) Escape latency (hidden platform). (B) Swimming speed (hidden platform). (C) Representative swimming tracks (probe test). (D) Platform-crossing number (probe test). (E–F) Distance traveled and time in the target quadrant (probe test). (G–H) Escape latency and swimming speed (visible platform). The results are presented as the mean ± standard deviation (SD). n = 6. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus. the CON group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the CIH group, ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus the SDD group.

Fig. 10.

An illustration of the potential neuroprotective mechanism of SDD on CIH-exposed HT22 cells. SDD alleviates CIH-induced neuronal damage by improving mitochondrial dysfunction via activation of the IGF1/IGFR signaling pathway.

4. Discussion

CIH exposure leads to hippocampal neuronal damage,³⁸ and repeated cycles of hypoxia/reoxygenation lead to decreased synaptic plasticity and memory. Exposure to CIH for only 7 days has been shown to reduce cell activity and synaptic plasticity in the hippocampus of rats.³⁹ A previous study showed that IGF1 has potent neuroprotective effects on promoting neuronal survival, differentiation, and hypoxic repair.⁴⁰ IGF1 binding to IGFR prevents neuronal cell damage and apoptosis caused by ischemia and hypoxia.⁴¹ A study showed that CIH exposure decreased serum IGF1 concentration and inhibited the growth of premature rats.⁴² Our outcomes suggested that the expression of IGF1 and IGFR peaked after 12 h of CIH exposure, which may be a compensatory mechanism to help neurons survive. To resist repeated hypoxia/reoxygenation-induced oxidative stress and ROS-induced apoptosis, hippocampal neurons will express more IGF1 to alleviate cell damage and promote neuronal repair. The expression of IGF1 and IGFR decreases at 24 and 48 h under CIH exposure, which may be related to neuronal damage. Thus, CIH exposure for an extended period of time will overwhelm the compensatory mechanism,⁴⁰ leading to a sharp decrease in the expression levels of IGF1 and IGFR.

Mice lacking IGF1 exhibit high postnatal mortality, growth retardation, infertility and severe defects in the development of major organ systems.⁴³ A previous study showed that IGF1 administration before noise exposure in adult rats resulted in significant improvements in hearing and HC impairment.⁴⁴ Saber⁴⁵ showed that treatment of infarcted rats with exogenous IGF1 after stroke reduced the size of their cerebral infarcts and significantly improved sensorimotor function. IGF1 has pleiotropic effects on neurons including migration to axons, axonal growth, and neuronal survival.^46,47 To investigate the effects of IGF1 on neurons, in this study, HT22 cells were exposed to various concentrations of exogenous IGF1 under CIH conditions. The results showed that the cell viability was significantly decreased after CIH exposure, but was significantly increased after administration of 0.1, 0.3, or 1 μg/mL IGF1 compared to the CIH group. These data showed that exogenous administration of IGF1 improved the decreased viability of HT22 cells caused by CIH exposure. It is well known that mitochondria directly affect neuronal survival and function.⁴⁸ Hypoxia can cause mitochondrial metabolism disorders and oxidative stress, resulting in decreased ATP production and mitochondrial respiratory chain enzyme activity, and production of ROS.⁴⁹ Free radicals hinder axonal transport, cause neuroinflammation, and interfere with mitochondrial activity, which contribute to the onset and progression of cognitive deficits. The loss of neurons in neurodegenerative disorders such as Alzheimer's disease (AD) is influenced by all of these variables. Indeed, many neurodegenerative disorders have mitochondrial dysfunction as part of their pathophysiology, which plays an important role in neuronal damage and cognitive function.⁵⁰ Oxidative stress induces mitochondrial DNA mutations, alters calcium homeostasis, interrupts the respiratory chain in the mitochondria, and changes the permeability of the mitochondrial membrane. Through oxidative phosphorylation, mitochondria produce more vital chemicals, adapt to oxidative stress, and supply cells with ATP. Oxidoreductase is an enzyme that catalyzes the transfer of electrons from the electron donor to the electron acceptor along the inner mitochondrial membrane. When this process is inefficient, ROS-generating mitochondrial mutations can be produced mutations and ROS production can lead to neuronal dysfunction.⁵¹ General impairment of oxidative phosphorylation affects the excitability and maturation of neurons by altering the expression of sodium channels and ion transporters, thereby causing poor maintenance of neuronal ion gradients and disrupting intracellular ion homeostasis.^52,53 ERK is extremely sensitive to oxidative stress.⁵⁴ When ERK is active, the CREB transcription factor can be phosphorylated at serine 133, resulting in activated transcriptional complexes that transcribe target genes.⁵⁵ CREB is crucial for neuronal survival and development. BDNF, a downstream target of CREB, increases mitochondrial ATP synthesis by increasing respiratory coupling, which supports neuroprotective processes.⁵⁶ Decreased BDNF expression may promote brain aging by disrupting a variety of complex physiological processes, including mitochondrial disorders, energy metabolism, and oxidative stress.⁵⁷ The expression of PSD-95 in the brain is decreased when the level of oxidative stress in mitochondria is increased.⁵⁸ Activation of the ERK/CREB/BDNF pathway can reduce mitochondrial oxidative stress. Importantly, phosphorylation of ERK and CREB is associated with long-term potentiation and memory.⁵⁹ CREB phosphorylation regulates BDNF expression, which is essential for neuronal growth and plasticity.60, 61, 62, 63, 64 PSD-95 participates in the trafficking of postsynaptic receptor channels and is affixed to the cytoplasm below the postsynaptic membrane.⁵ The ERK/CREB/BDNF signaling pathway is neuroprotective and regulates synaptic plasticity and memory capacity.⁶⁵

IGF1 reduces free radical production and oxidative damage, and increases ATP production. The cytoprotective effects of IGF1 are closely related to mitochondria. IGF1 can improve mitochondrial dysfunction.⁶⁶ IGF1 has pleiotropic effects and is essential for neural plasticity, neuronal survival, and stimulation of hippocampal neurogenesis in the brain.⁶⁷ Previous studies have shown that conditional deletion of dopamine neuron-derived IGF1 in adult mice results in reduced dopamine levels in the striatum and defects in dopamine neuron firing, leading to reduced spontaneous movements and impaired exploratory and learning behaviors.⁶⁸ In the adult mouse brain, IGF1 regulates neuronal plasticity and actively promotes neurogenesis.⁶⁹ IGF1 promotes neurite outgrowth⁷⁰ and protects cells from mitochondrial-level apoptotic stimuli.⁷¹ Our results suggested that exogenous IGF1 has a protective effect on CIH-induced neuronal damage. The mechanism is closely related to improving mitochondrial dysfunction and reducing the level of oxidative stress.

Our previous study indicated that CIH-exposed mice showed signs of Qi and Yin deficiency.⁵ SDD is composed of Shengmai San and Liuwei Dihuang Decoction, consistent with the basic pathogenesis of Qi and Yin deficiency caused by CIH. Shengmai San inhibited apoptosis of hippocampal neurons and significantly improved the learning memory ability of VD rats.⁷² Long-term administration of Liuwei Dihuang Decoction and its active ingredients enhanced LTP in the hippocampal tissues of animals.⁷³ And Liuwei Dihuang Decoction can correct abnormal gene expression in the hippocampus of senescence-accelerated mice, improve learning and memory, and inhibit neuronal damage. The mechanism is closely related to the activation of the IGF1/IGFR pathway by a kidney-tonifying TCM.²⁶ Catalpol, the main active ingredient of Rehmannia,⁷⁴ can promote cell survival by regulating IGF1.⁷⁵ Ginsenosides can improve hypoxia-induced vascular injury by activating the IGF1/IGFR signaling pathway.⁷⁶ Our study showed that SDD increased IGF1/IGFR expression under CIH conditions, indicating that SDD may alleviate neuronal damage and promote cell survival by increasing the expression of IGF1.

The interaction between IGF1 and IGFR activates two major signaling pathways, PI3K/AKT/GSK3β and MAPK.⁷⁷ Recent studies have shown that catalpol has protective effects against several neurovascular diseases. Catalpol decreases GSK3β expression by activating the PI3K/AKT and MAPK/ERK1/2 signaling pathways⁷⁸ and promotes neurogenesis. As a downstream signaling molecule of AKT, GSK3β plays an important role in neurodegenerative diseases. Ginsenoside Rg1 can reduce tau hyperphosphorylation in dementia rats by inhibiting GSK3β expression.⁷⁹ In addition, long-term treatment with Rg1 can improve synaptic plasticity in AD rats and may increase the expression of phosphorylated BDNF and CREB by upregulating the BDNF/TrkB. Ophiopogonis can upregulate the phosphorylation levels of PI3K, AKT, ERK1/2, BDNF, and CREB to promote neuronal survival and neurite growth⁸⁰ Schisandra chinensis Fructus increases the protein levels of BDNF and PSD-95 in the hippocampus by activating the CREB/ERK and PI3K/AKT/GSK3β signaling pathways, and exerts antidepressant-like effects on chronic unpredicted mild stress.⁸¹

In this study, siRNA-mediated knockdown of IGF1 and the IGFR antagonist AG1024 both inhibited activation of the PI3K/AKT/GSK3β and RAS/RAF/MAPK pathways, and also reversed the effects of SDD treatment on neuronal damage and mitochondrial damage under CIH conditions. These data suggest that SDD acts on the key target of IGF1 to alleviate CIH-induced neuronal damage. The results of this study showed that SDD increased the expression levels of IGF1, IGFR, and IGFR downstream signaling molecules PI3K/AKT/GSK3β and RAS/RAF/MAPK under CIH conditions. Our study indicates that SDD can alleviate CIH-induced neuronal damage through the IGF1/IGFR signaling pathway.

5. Conclusion

In conclusion, this study demonstrated that CIH exposure caused neuronal damage by downregulating IGF1/IGFR expression. SDD alleviated CIH-induced neuronal damage by improving mitochondrial dysfunction via activation of the IGF1/IGFR signaling pathway. Furthermore, our findings provide new insights for treating of OSA-related cognitive dysfunction in TCM.

Disclosure

The authors report no conflicts of interest in this work.

Author Contributions

Shengchang Yang and Ensheng Ji conceived and designed the experiments. Xue Chen, Kerong Qi, and Jianchao Si mainly performed the experiments and wrote the paper. Dongli Li, Xintong Fan, and Mengfan Sun analyzed the data and contributed to reagents and materials.

Abbreviations

OSA	Obstructive sleep apnea
CIH	Chronic intermittent hypoxia
TEM	Transmission electron microscopy
TCM	Traditional Chinese Medicine
SDD	Shengmai Dihuang Decoction
AD	Alzheimer's disease
IGF1	Insulin-like growth factor 1
IGFR	Insulin-like growth factor 1 receptor
BDNF	Brain-derived neurotrophic factor
PSD-95	Postsynaptic density protein 95
MMP	Mitochondrial Membrane Potential
P-CREB	Phosphorylated cAMP-response element binding protein
CREB	cAMP-response element binding protein
RAF	Raf-1 proto-oncogene, serine/threonine kinase
ERK	Extracellular regulated protein kinases
P-ERK	Phosphorylated extracellular regulated protein kinases
RAS	Rat sarcoma
MAPK	Mitogen-activated protein kinase
PI3K	Phosphoinositide-3- kinase
AKT	V-akt murine thymoma viral oncogene homolog 1
P-AKT	Phosphorylated v-akt murine thymoma viral oncogene homolog 1
GSK3β	Glycogen synthase kinase 3β
MAP2	Microtubule-associated protein 2
ILPs	Insulin-like peptides
ATP	Adenosine Triphosphate
ROS	Reactive oxygen species

Funding

This work was supported by the National Natural Science Foundation of China (82274617), the Hebei Natural Science Foundation (H2022423352, H2022423370), the Central Leading Local Science and Technology Development Fund Project (216Z7704G), the Science and Technology Research Fund Project of Hebei Colleges and Universities (ZD2020142).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article.