Tetrandrine mediates autophagy via sirtuin 3/adenosine 5-monophosphate-activated protein kinase/mammalian target of rapamycin signal pathway to attenuate early brain injury after subarachnoid hemorrhage

Abstract

Objective

Early brain injury (EBI) is the main cause of poor outcomes in patients with subarachnoid hemorrhage (SAH). Tetrandrine (Tet) is the root of Stephania tetrandra S Moore extract that has been shown to promote neuronal survival and regulate a variety of signaling pathways; however, the mechanism through which it exerts neuroprotective effects in patients with SAH is unknown. This investigation was to examine Tet’s effect on EBI in SAH rats.

Basic Methods

We divided the rats into four groups. The effects of Tet treatment on the pathological changes of neurons in rat brains were evaluated, as well as autophagy-related and signaling pathway proteins.

Main Results

We found that Tet had a neuroprotective effect on EBI after SAH, as evidenced by the fact that Tet ameliorated SAH-mediated neurologic impairment and neuronal morphological damage and reduced brain water content, neuronal apoptosis rate, and neuronal cell loss. Tet decreased the LC3II/LC3I ratio, elevated P62 protein expression, and inhibited autophagosome production after SAH. Tet may have increased sirtuin 3 (SIRT3) expression, decreased adenosine 5-monophosphate-activated protein kinase (AMPK) phosphorylation, and increased phosphor–mammalian target of rapamycin (mTOR) levels, all of which may have occurred particularly via SIRT3/AMPK/mTOR signaling pathway activation; However, this trend can be reversed by 3-(1H-1,2,3-triazol-4-yl) pyridine (SIRT3 inhibitors).

Conclusions

Tet exerts neuroprotective effects by inhibiting autophagy, this may be associated with SIRT3’s inhibitory effect on the AMPK/mTOR signaling pathway. This inhibition could function as a potential mechanism for the neuroprotective effects observed in patients suffering from SAH.

Introduction

Subarachnoid hemorrhage (SAH) is a stroke with a poor prognosis. Despite accounting for just around 5% of all strokes, SAH has a mortality rate of 45–50%. Around 30% of survivors experience mild to severe disability [1]. For many years, it was believed that delayed cerebral vasospasm was the most important factor influencing the outcome of patients with SAH. According to some studies, even lowering delayed cerebral vasospasm following SAH has been shown to not affect the prognosis of patients with SAH [2]. Thus, numerous studies have mainly focused on SAH-induced early brain injury (EBI). EBI is a term used to describe a series of pathophysiological processes that occur during the first 72 h after SAH. These processes include intracranial pressure elevation, reduced blood flow to the cerebral, decreased pressure of cerebral perfusion, activated autophagy, blood–brain barrier disruption, inflammation, oxidative stress, death of neuronal cells, and cerebral edema [3]; however, the role of autophagy in EBI is unclear, it is indisputable that EBI activates autophagy [4]. But autophagy is a protective mechanism or a mechanism of damage in EBI is still a topic of debate. Resveratrol can reduce EBI by activating autophagy. In contrast, the use of melatonin to inhibit autophagy also serves the purpose of reducing EBI [5,6].

At 24 h after SAH, SAH inhibits recombinant sirtuin 3 (SIRT3) protein expression [7]. Puerarin increases the expression of SIRT3 and reduces EBI in the brain [8]. Other studies have supported this idea. One study reported the role of SIRT3 in neuronal ischemia [9]. In that study, the researchers upregulated the protein expression of SIRT3 using lentivirus transfection. The results indicated that SIRT3 could significantly reduce neuronal apoptosis and may exert neuroprotective effects by autophagy activation via the signaling pathway involving the adenosine 5-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR); however, it is not known whether and how SIRT3 can regulate autophagy through the signaling pathway involving AMPK/mTOR in-vivo experiments in the SAH model.

Tetrandrine (Tet) is an alkaloid extracted from the root of Stephania tetrandra S Moore. Tet has many pharmacologic effects, including antiautophagic, antitumor, anti-inflammatory, and antioxidative stress effects [10]. It has been shown that Tet is a neuroprotective agent against ischemia–reperfusion (I/R) injury via inhibiting the activation of the NLRP3 inflammasome via Sirt-1 upregulation [11]. Meanwhile, Tet triggered autophagy in bladder cancer cells of humans via a signaling pathway involving AMPK/mTOR [12]; however, no reports have explored the neuroprotective effect of Tet on EBI.

The purpose of the current work was to determine whether Tet could regulate autophagy via the SIRT3–AMPK–mTOR signaling pathway and consequently improve EBI after SAH.

Materials and methods

Animals

Sprague–Dawley rats (male; 300–330 g) were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats were housed in groups of five in cages with natural daylight at 23 ± 1 °C and 50% relative humidity and provided unrestricted access to appropriate water and a proper diet. To accustom the laboratory environment, the Sprague–Dawley rats were adaptively fed for 1 week. All experimental protocols were according to the regulations for laboratory animal management issued by the People’s Republic of China’s Ministry of Science and Technology (1988) no. 134. The Animal Care and Use Committee at Hebei General Hospital in Shijiazhuang, China, approved all animal research, which was carried out in compliance with ethical standards.

Rat subarachnoid hemorrhage model

As previously described, experimental SAH was induced utilizing a model of endovascular perforation [13]. Pentobarbital (40 mg/kg) was used to anesthetize the rats intraperitoneally. The rats were put under anesthesia and placed on the operating table in the supine position, and the area surrounding the incision was sterilized by shaving. The carotid artery on the right side was exposed, along with its branches. A suture of 4-0 nylon was put into the right internal carotid artery from the external carotid artery. When the nylon wire encountered resistance, it was advanced further 4 mm, puncturing the middle cerebral artery and the anterior arterial bifurcation. In the sham operation, the nylon wire was retracted when resistance was encountered.

Drug administration and experimental design

Tet (TCI, Tokyo, Japan) and 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP; MedChemExpress, Monmouth Junction, New Jersey, USA) were dissolved in dimethyl sulfoxide (DMSO; <2%). Four groups of rats were randomly assigned: (a) sham group: equal volumes of vehicle (DMSO < 2%) were intraperitoneally injected into rats after the sham operation. (b) SAH group (SAH): rats were immediately injected intraperitoneally with an equal volume of vehicle after SAH surgery. (c) Tet-treated SAH model group (Tet): rats were immediately injected intraperitoneally with Tet (20 mg/kg) after SAH surgery [14]. (d) 3-TYP and Tet-treated SAH model group (Tet + 3-TYP): rats were pretreated with a 30 mg/kg intraperitoneal injection of 3-TYP and were subsequently administered 3-TYP once every 2 days three times in total before SAH surgery [15]. The rats were then immediately treated with Tet (20 mg/kg) through intraperitoneal injection following SAH surgery. The does and dissolution methods of Tet and 3-TYP were determined by pre-experiment and described previously.

Subarachnoid hemorrhage grade

Previously established grading scales were used to quantify the severity of SAH [16]. The basal cistern was separated into six sections. The SAH severity was assessed on a scale ranging from 0 to 3, with the following: No SAH in grade 0; low subarachnoid blood in grade 1; clots in identified arteries in grade 2; major hemorrhage overlying cerebral arteries in group 3. The total SAH score for the final region was the sum of the total SAH scores for the six areas. The SAH grade was calculated blindly. Animals with mild SAH (SAH grade ≤ 8) were excluded.

Neurologic score

The Garcia scale system was used to determine the neurologic score, as discussed previously [17]. The investigation consisted of six tests, each of which could be scored from 0 to 3 or 1 to 3, and they were as follows: spontaneous activity, four-limb movement symmetry, outstretching of the forepaw, proprioception of the body, climbing, and vibrissae touch response. The range of possible scores was from 3 to 18. The neurologic function score assessments are blinded.

Brain water content

The brain water content was determined in the manner previously described [18]. Rats were anesthetized deeply and sacrificed 24 h following SAH. The brains were promptly removed, preserving only the right hemispheres. Weighing the right hemispheres yielded the wet weight, which was subsequently dried in an oven at 105 °C for 24 h. The formula shown below was utilized to compute brain water content:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{B}\mathrm{r}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{w}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}=\frac{\mathrm{w}\mathrm{e}\mathrm{t}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{d}\mathrm{r}\mathrm{y}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}{\mathrm{w}\mathrm{e}\mathrm{t}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}\times 100.}\end{array}}$$

Staining with Nissl and hematoxylin and eosin

The rats were completely anesthetized and euthanized following a 24-h period of SAH, and brain tissue was extracted. The brain tissue was fixed with 4% paraformaldehyde for 24 h and then immersed in paraffin. The brain tissue was cut into thick slices of 5-μm using a tissue Slicer (RM2016; Leica Instrument Shanghai Ltd, Shanghai, China). The sheets were dewaxed and stained with a preheated toluidine blue aqueous solution (0.5%) to achieve the Nissl staining. Hematoxylin and eosin were used for staining. Brain tissue sections were examined and photographed under a microscope. Nissl-positive cells (number of intact neurons) were analyzed using ImageJ 1.37 image analysis software (Bethesda, Maryland, USA).

Terminal deoxynucleotidyl transferase dUTP nick end labeling analysis

Sections of paraffin were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to identify apoptosis in each group of samples. We performed the method based on the instructions in the TUNEL assay kit (Servicebio, Wuhan, China). Each paraffin section (thickness = 5 μm) was deparaffinized and rehydrated with xylene and an alcohol gradient. After that, the sections were incubated for 20 min by proteinase K at a temperature of 37 °C, and then the sections were three times washed with PBS. The sections were incubated at room temperature in a 3% H₂O₂ solution for 20 min, protected from light, and then three times washed the sections with PBS. Slices were put into the buffer to fully cover the tissue and then incubated at room temperature for 10 min. Brain sections were stained with 3,3′-diaminobenzidine after treatment with a TUNEL reaction mixture. The number of cells positive for TUNEL was evaluated by counting the five fields without random crossover. The level of neuronal damage was determined by computing the apoptosis rate (percentage of the number of positive cells in the total number of cells). We evaluated the data using the image analysis software ImageJ 1.37.

Transmission electron microscopy assay

Autophagy was assessed at 24 h following SAH by transmission electron microscopy. Briefly, the rats were given a general anesthetic and then rapidly euthanized. The brain was removed, and the right cerebral cortex was trimmed into 1-mm³ blocks using a scalpel. Tissue was incubated in 4% glutaraldehyde overnight at 4 °C. The following procedures were performed in the manner indicated previously [19]. After the tissue was cut into ultrathin sections, the number of autophagosomes was observed by electron microscopy.

Immunofluorescence staining

After dewaxing and rehydration, the brain sections were microwaved for repair three times and immersed in 3% H₂O₂ for 25 min. Sections were blocked with BSA for 30 min and then treated with anti-SIRT3 rabbit polyclonal antibody overnight (1 : 200; Abclonal, Wuhan, China) at 4 °C. After washing away the primary antibody with PBS, the section was incubated with goat anti-rabbit immunoglobulin G fluorescent secondary antibody for 50 min (1 : 1000; Servicebio, Wuhan, China). An antiquenching agent was applied to the sections 10 min after 4',6-diamidino-2-phenylindole staining. We observed the tissue sections with a laser scanning confocal microscope under ×400 magnification. We used Image Pro software to calculate the fluorescence intensity.

Western blotting

The supernatant was extracted by homogenization and centrifugation of the ipsilateral cortex at 4 °C. The total proteins (25 μg) were separated by SDS–polyacrylamide gel electrophoresis. The proteins were transferred onto a membrane typically of polyvinylidene fluoride, after electrophoresis. The membrane was blocked in nonfat dried milk (5%) for 2 h. The membrane was treated with the primary antibodies listed below for an overnight period at 4 °C: LC3 (1 : 1000; Sigma, Louis, Missouri, USA), SIRT3 (1 : 1000; Abclonal, Wuhan, China), P62 (1 : 1000; Servicebio, Wuhan, China), AMPK (1 : 1000; Cell Signaling Technology, Danver, Massachusetts, USA), P-AMPK (1 : 1000; Cell Signaling Technology, Danver, Massachusetts, USA), mTOR (1 : 1000; Cell Signaling Technology, Danver, Massachusetts, USA), P-mTOR (1 : 1000; Cell Signaling Technology, Danver, Massachusetts, USA), β-actin (1 : 1000;Servicebio, Wuhan, China). Then, appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 5000; Servicebio, Wuhan, China) were chosen to be incubated with the membranes at room temperature for 2 h. The membrane was washed, developed via chemiluminescence, exposed, and photographed. The band densities were quantified with ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Statistical analysis

The data are presented as mean ± SD. GraphPad Prism 8.0 (GraphPad Software,San diego, California, USA) was employed to conduct the statistical analysis. Comparisons of multiple groups were conducted using a one-way analysis of variance; Dunnett or Tukey tests were utilized for the post hoc test. P less than 0.05 was regarded as significantly different.

Results

Mortality and subarachnoid hemorrhage grade

A total of 59 rats were used; 47 rats underwent SAH induction, 12 rats received sham surgery. Four rats were excluded because their SAH grade was low (SAH grade ≤ 8). The mortality of all SAH rats was 16.27% (7/43) (Table 1). In the sham group, there were no bleeding spots or clots in the skull base brainstem or Willis ring. In the SAH group and Tet group, clots were observed overlying the vessels in both the Willis ring and brainstem. Some of the vessels were not identifiable because of clot occlusion (Fig. 1a). After scoring, it was observed that the sham and SAH groups had a statistically significant difference, as depicted in Fig. 1b (P < 0.01), in the Tet group. In comparison, no statistically significant difference in SAH scores was identified between the SAH and Tet groups (P > 0.05; Fig. 1b).

Table 1  .

Summary of animal usage and mortality

Groups	Mortality	Exclusion
Sham	0 (0/12)	0
SAH	20% (3/15)	1
Tet	14.28% (2/14)	2
3-TYP	14.28% (2/14)	1

3-TYP: 3-(1H-1,2,3-triazol-4-yl) pyridine; SAH, subarachnoid hemorrhage; Tet, tetrandrine.

Fig. 1.

Effect of Tet on SAH score, brain water content, and neurologic function at 24 h after SAH. (a) A representative brain sample. (b) The SAH grades of all groups (n = 12). (c) Brain water content quantification (n = 4). (d) Neurologic score quantification (n = 4). The data are presented as mean ± SEM. **P < 0.01 vs. SAH, ^##P < 0.01 vs. sham. SAH, subarachnoid hemorrhage; Tet, tetrandrine.

Tet significantly reduced neurologic deficits and brain edema following subarachnoid hemorrhage

Brain edema and neurologic function were examined 24 h following SAH to determine the effect of Tet on EBI. In comparison to the sham group, the SAH group’s brain water content increased considerably, and the neurologic deficit scores decreased significantly, as presented in Fig. 1c and d (P < 0.01). Moreover, when the Tet group was compared with the SAH group, we noticed that the Tet group’s brain water content reduced dramatically and the neurologic deficit score increased significantly (P < 0.01; Fig. 1c and d).

Tetrandrine attenuated neuronal death at 24 h after subarachnoid hemorrhage

To more accurately examine Tet’s effect on EBI following SAH, we employed TUNEL and Nissl staining to determine Tet’s effect on neurons 24 h after SAH. As illustrated in Fig. 2, the findings of Nissl staining revealed that the sham group’s neuronal cells were morphologically diverse and neatly arranged, with deep staining of Nissl bodies. The number was also normal, and in the SAH group, the cortical area on the SAH side of the rats was severely damaged, with a much-disorganized arrangement and increased intercellular space, accompanied by a large loss of Nissl-positive cells (normal neurons; P < 0.01; Fig. 2a and b); however, in comparison with the SAH group, the damage to the cortical area on the SAH side of the group was greatly alleviated, and the number of neuronal cells was increased (P < 0.01; Fig. 2a and b). The results of staining of TUNEL demonstrated that rat cerebral cortical apoptotic cells in the SAH group were severely stained with brown apoptotic cells, and the apoptotic rate was dramatically raised in comparison to the sham group (P < 0.01; Fig. 2c and d). The apoptotic rate of cells in the cerebral cortical area of the Tet group was conspicuously lower than that of the SAH group (P < 0.01; Fig. 2c and d). These results suggest that Tet could have a neuroprotective effect on EBI.

Fig. 2.

Tet reduces early brain damage following SAH. (a) Representative images of the sections stained with Nissl (×200; n = 4). The black arrows indicate damaged neurons. (b) Number of Nissl-positive cells. (c) Representative images of TUNEL-stained sections (×200; n = 4). The black arrows indicate apoptotic cells. (d) Apoptosis rate quantification. The data are presented as mean ± SEM. **P < 0.01 vs. SAH, ^##P < 0.01 vs. sham. SAH, subarachnoid hemorrhage; Tet, tetrandrine; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Tetrandrine inhibited subarachnoid hemorrhage-induced autophagy

We also explored the effect of Tet on autophagy in SAH. The SAH group had a greater number of autophagosomes than the sham group (P < 0.01; Fig. 3c, d, and f). In comparison to the SAH group, the Tet group demonstrated a substantial decrease in autophagosomes (P < 0.01; Fig. 3d–f). In addition, we measured the expression of two important autophagy biomarkers (Figs. 3a and S1a and b, Supplemental Digital Content 1, https://links.lww.com/WNR/A826). Western blotting revealed an increase in LC3II/LC3I in contrast to the sham group (P < 0.01, Fig. 3b) and a reduction in P62 expression in the SAH group (P < 0.01; Fig. 3b). In comparison to the SAH group, the Tet group had a lower LC3II/LC3I ratio (P < 0.01; Fig. 3b) and a higher level of P62 expression (P < 0.01; Fig. 3b). These findings revealed that Tet may inhibit autophagy activated by EBI.

Fig. 3.

Effects of Tet regulates autophagy following SAH. (a) P62 and LC protein expression were evaluated through Western blotting. (b) Quantified protein bands of P62 with β-actin and LC3-II with LC3-I as control (n = 4). (c–e) Representative images of transmission electron microscopy. (f) Number (red arrows) in neurons was calculated (n = 3, scale bar = 5 μm). The data are expressed as mean ± SEM. ^##P < 0.01 vs. sham, **P < 0.01 vs. SAH. LC, light chain; SAH, subarachnoid hemorrhage; Tet, tetrandrine.

Tetrandrine regulates sirtuin 3 and autophagy

We used 3-TYP (SIRT3 inhibitor) to demonstrate that Tet can activate SIRT3 and inhibit autophagy. The results are presented in Figs. 4 and 5. We explored the SIRT3 protein by immunofluorescence and Western blot (Figs. 4a, 5a, and S2a, Supplemental Digital Content 1, https://links.lww.com/WNR/A826). Our results demonstrated that, in comparison to the sham group, both the fluorescence intensity and protein expression levels of SIRT3 in the cerebral cortex of rats in the SAH group were significantly decreased, as depicted in Fig. 4b (P < 0.01) and Fig. 5b (P < 0.05). The Tet group exhibited higher protein expression and fluorescence intensity compared with the SAH group, as illustrated in Fig. 4b (P < 0.01) and Fig. 5b (P < 0.05). Protein expression and fluorescence intensity were significantly lower in the Tet + 3-TYP group than in the Tet group, as shown in Fig. 4b (P < 0.01) and Fig. 5b (P < 0.05). Furthermore, we discovered that the LC3II/LC3I ratios were significantly higher in the SAH group in comparison to the sham group (P < 0.01; Figs. 5a, c, and S2b, Supplemental Digital Content 1, https://links.lww.com/WNR/A826). When compared with SAH, the Tet group had considerably lower LC3II/LC3I ratios (P < 0.01; Figs. 5a, c, and S2b, Supplemental Digital Content 1, https://links.lww.com/WNR/A826). The LC3II/LC3I ratios were considerably higher in the Tet + 3-TYP group than in the Tet group (P < 0.01; Figs. 5a, c, and S2b, Supplemental Digital Content 1, https://links.lww.com/WNR/A826).

Fig. 4.

Effects of Tet on the cells labeled with SIRT3 in the cortical areas of the brain. (a) Representative immunofluorescence images of SIRT3 (n = 4, ×200). (b) Analysis of fluorescence intensity. The data are presented as mean ± SEM. *P < 0.05 vs. SAH, **P < 0.01 vs. SAH, ^#P < 0.05 vs. sham, ^##P < 0.01 vs. sham, ^$P < 0.05 vs. Tet, ^$$P < 0.01 vs. Tet. SAH, subarachnoid hemorrhage; SIRT3, sirtuin 3; Tet, tetrandrine.

Fig. 5.

Effects of Tet regulate SIRT3, autophagy, and AMPK/mTOR signaling pathway. (a) SIRT3, LC3, AMPK, P-AMPK, mTOR, and P-mTOR protein expression were evaluated through Western blotting. (b and c) Quantified protein bands of SIRT3 with β-actin and LC3-II with LC3-I as control (n = 4). (d–g) Quantified protein bands of AMPK, P-AMPK, mTOR, and P-mTOR protein levels with β-actin as control in each group (n = 4). (h and i) Quantified protein bands of P-AMPK with AMPK and P-mTOR with mTOR as control (n = 4). The data are presented as mean ± SEM. *P < 0.05 vs. SAH, **P < 0.01 vs. SAH, ^#P < 0.05 vs. sham, ^##P < 0.01 vs. sham; ^$P < 0.05 vs. Tet, ^$$P < 0.01 vs. Tet. AMPK, adenosine 5-monophosphate-activated protein kinase; LC, light chain; mTOR, mammalian target of rapamycin; SAH, subarachnoid hemorrhage; SIRT3, sirtuin 3; Tet, tetrandrine.

Involvement of the adenosine 5-monophosphate-activated protein kinase– mammalian target of rapamycin pathway in sirtuin 3-induced neuroprotection

To further address that Tet-induced neuroprotection was mediated through SIRT3/AMPK/mTOR signal pathway, we analyzed AMPK, P-AMPK, mTOR, and P-mTOR expression using Western blot (Figs. 5a, S3 g1, g2, g3, g4, and S4 g1, g2, g3, g4 Supplemental Digital Content 1, https://links.lww.com/WNR/A826). We discovered that there was no disparity in AMPK expression among the groups (P > 0.05; Fig. 5d) compared with the sham group, the expression level of P-AMPK protein in the SAH group increased (P < 0.05; Fig. 5e), its levels were reduced following Tet treatment (P < 0.05; Fig. 5e), in comparison to the Tet group, the Tet + 3-TYP group had a higher level of P-AMPK expression (P < 0.05; Fig. 5e). The protein quantity of mTOR in the SAH group was lower than that in the sham group (P < 0.05; Fig. 5f); however, the expression levels of mTOR protein in the SAH group and the Tet group were similar (P > 0.05; Fig. 5f) and, the mTOR protein content in the Tet + 3-TYP group was higher than that in the Tet group (P < 0.05; Fig. 5f). The amount of P-mTOR protein in the SAH group was significantly reduced compared with the sham group (P < 0.05; Fig. 5g), in the Tet group, the level of P-mTOR protein showed a significant increase when compared to the SAH group (P < 0.05; Fig. 5g); however, the protein expression levels of P-mTOR in the Tet group and the Tet + 3-TYP group were similar (P > 0.05; Fig. 5g).

To observe the activation of the SIRT3–AMPK–mTOR signaling pathway, we analyzed the ratios of P-AMPK to AMPK and P-mTOR to mTOR in each group. We observed that the P-AMPK/AMPK ratios were considerably higher in the SAH group in comparison to the sham group (P < 0.01; Fig. 5h). P-AMPK/AMPK ratios were dramatically lowered in the Tet group in comparison to the SAH group (P < 0.01; Fig. 5h). The P-AMPK/AMPK ratios were considerably higher in the Tet + 3-TYP group in comparison to the Tet group (P < 0.01; Fig. 5h). We also observed that the P-mTOR/mTOR ratio in the SAH group was considerably lower as compared with the sham group (P < 0.01; Fig. 5i). When compared with the SAH group, the Tet group had a considerably increased P-mTOR/mTOR ratio (P < 0.01; Fig. 5i); however, the P-mTOR/mTOR ratio decreased in the Tet + 3-TYP group in comparison to the Tet group, as shown in Fig. 5i (P < 0.01).

Meanwhile, based on the data presented in Fig. 6, under similar SAH score (Fig. 6b), We found that 3-TYP reversed the neuroprotective effect of Tet on SAH by increasing the brain water content and reducing the neurologic deficit scores in comparison to the Tet group, as shown in Fig. 6c and d (P < 0.05). In addition, the results of hematoxylin and eosin staining presented in Fig. 6a revealed that the morphology of neuronal cells in the rat cerebral cortex in the sham-operated group was normal. The presence of a significant number of neuronal cells in the rat cerebral cortex in the SAH group demonstrated pyknosis, deep staining, shrunken cell bodies, agglomerated cytoplasm, and a large number of vacuoles; the aforementioned condition in the brain tissue of rats in the Tet group was greatly alleviated than SAH group. In addition, the Tet + 3-TYP group’s neuronal cells were severely damaged in comparison to the Tet group and nuclear pyknosis and deeply stained nuclei were aggravated.

Fig. 6.

3-TYP eliminates the cerebral protective effect of Tet. (a) Representative images of HE-stained sections (×200; n = 4). (b) SAH grades of all groups (n = 12). (c) Quantification of neurologic score (n = 4). (d) Brain water content quantification (n = 4). The data are presented as mean ± SEM. *P < 0.05 vs. SAH, **P < 0.01 vs. SAH, ^#P < 0.05 vs. sham, ^##P < 0.01 vs. sham, ^$P < 0.05 vs. Tet, ^$$P < 0.01 vs. Tet. 3-TYP, 3-(1H-1,2,3-triazol-4-yl) pyridine; HE, hematoxylin and eosin; SAH, subarachnoid hemorrhage; Tet, tetrandrine.

Submission file

All the original pictures of Western blot were posted in Figs. S1–S4, Supplemental Digital Content 1, https://links.lww.com/WNR/A826.

Discussion

In the current work, we found that Tet had a protective effect on the brain of the rat during EBI following SAH. Tet can reduce brain edema, alleviated neuropraxia grade injury, improve pathological and morphological changes in the cerebral cortex, and decrease the apoptotic rate of brain cells. The protective function of Tet may be partly attributed to the inhibition of autophagy. At the same time, our study found that Tet upregulated the expression of SIRT3, which inhibits autophagy by suppressing the AMPK/mTOR signaling pathway. These findings show that Tet may be a protective agent that reduces brain damage in EBI after SAH.

In recent years, researchers have widely accepted the view that EBI plays an important role in patients with SAH. Within 72 h of SAH, pathophysiological alterations in the brain include intracranial pressure elevation, reduced blood flow to the cerebral and pressure of cerebral perfusion, blood–brain barrier disruption, acute vasospasm, autoregulation dysfunction, and brain edema [4]. The complex set of pathological processes involved in EBI after SAH has led to nimodipine being the only effective treatment for SAH at present [20]. Therefore, it is essential to find an effective treatment for SAH. Several studies have shown that Tet protects the heart, liver, and brain from I/R injury [11,21,22]; however, the effects of Tet on SAH have yet to be proven. We explored the effect of Tet on EBI after SAH. Tet has an advantage of low molecular weight, which facilitates its crossing the blood–brain barrier [10]. Indicating that this agent has the potential to be protective for SAH. In addition, Tet decreased I/R damage in the brain in a middle cerebral artery blockage mouse model by regulating GRP78, DJ-1, and HYOU1 protein expression at 24 h after stroke, indicating the neuroprotective effect of Tet [14]. In our research, we found that Tet decreased EBI at 24 h after SAH, as confirmed by a decrease in brain edema, neurologic impairment, abnormally stained nerve cells by Nissl staining, and apoptotic cells in the brain by TUNEL staining. These results suggest a protective role of Tet at 24 h after SAH; however, the mechanisms underlying the Tet-dependent protective effects on EBI remain obscure.

To understand how Tet can reduce neuron damage 24 h after SAH, we examined the expression of proteins associated with autophagy. Autophagy is a process that has been conserved throughout evolution in eukaryotic cells [9]. It can sequester the targeted cytoplasmic components and send them to the lysosome for digestion, which keeps cells active by degrading and recycling the targeted cytoplasmic components, such as damaged organelles and long-lived proteins. Under normal circumstances, autophagy is at a low level and performs many physiological functions in most cells of the brain, such as intracellular quality control, cell development and death, immune suppression, and antiaging mechanisms [4]; however, whether enhanced autophagy is beneficial or harmful to the brain remains controversial. It is generally accepted that moderate autophagy will play a beneficial role in neuroprotective effects. On the other hand, excessive and insufficient autophagy can cause damage to cells [23]. The highest level of SAH-mediated autophagy in brain tissue was observed at 24 h after EBI; however, whether upregulation of autophagy is part of the treatment of EBI remains controversial. Specifically, resveratrol can reduce EBI by activating autophagy and inhibiting apoptosis. But, interestingly melatonin can also inhibit autophagy by reducing reactive oxygen species (ROS) levels and inhibiting MST1 protein cleavage. The same could be achieved to improve EBI [5,6]. This suggests that the relationship between autophagy and SAH has not been elucidated.

Autophagy begins with the formation of autophagosomes, and microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is necessary for the formation of autophagosomes. Therefore, LC3 can reflect the activity of autophagy. To expose the glycine residue and generate LC3I, the LC3 c-terminal polypeptide is degraded by ATG4 protease. LC3I covalently binds to phosphatidylethanolamine and interacts with, ATG12-ATG5-ATG16L, ATG3, and ATG7 complexes to produce LC3II. The expression level of the LC3II protein can reflect the number of autophagosomes. Autophagic activity can also be identified by detecting the conversion of LC3I to LC3II (LC3II/LC3I) [24]. p62 is an important bridge and adaptor protein in the signal transduction pathway and is involved in the formation of many protein complexes in cells. p62 has multiple structural domains, and different structural domains interact with corresponding proteins to mediate a variety of cellular functions including cellular autophagy [25]. P62 can bind to LC3 via the LC3 interacting region (LIR), and then the LC3 interacting region binds to ATG8. This is also necessary for autophagic degradation. p62 is wrapped by autophagosomes during autophagosome formation and eventually degraded via lysosomes. Therefore, P62 content decreases when autophagy is activated [26]. In the current investigation, we showed that SAH increased the LC3-1/LC3-2 ratio and decreased the expression level of p62, but Tet reversed this trend. At the same time, in addition, we observed that the Tet group had fewer autophagosomes than the SAH group. This shows that Tet’s neuroprotective role in SAH might well be associated with autophagy inhibition. A prior study showed that Tet induced death by activating autophagy in cancer cells [27]. This is different from our findings, and we hypothesize that the difference could be caused by the variation between tumor cells and normal nerve cells.

SIRT3 is mainly found in mitochondria and may play a protective role by scavenging ROS under pathophysiological conditions such as oxidative stress and metabolic disorders [28]. Overexpression of SIRT3 by lentiviral transcription reduces H₂O₂-mediated neural injury, and overexpression of SIRT3 regulates mitochondrial Ca²⁺ homeostasis and mitochondrial biogenesis, thereby protecting cortical neurons [29], which indicate that SIRT3 reduces oxidative stress by regulating mitochondrial function. The similarity is that cortical nerve cells were found to contribute to mitochondrial dysfunction in EBI after SAH [30]. Puerarin can upregulate the protein expression level of SIRT3, reduce ROS production, and improve EBI at 24 h after SAH [8]. In addition, melatonin can also upregulate SIRT3 production and exert neuroprotective effects in SAH [31]. The above study showed that EBI can be attenuated by pharmacologic upregulation of SIRT3 in EBI after SAH, which is similar to our findings. Our experimental results show that Tet can attenuate brain damage of SAH by upregulating SIRT3 expression and inhibiting autophagy; however, the SIRT3 inhibitor 3-TYP reversed these effects, which is similar to the findings of a previous study [32].

SIRT3 can regulate autophagy by multiple signaling pathways [33]. As for one of the clues, our results suggest that Tet-mediated upregulation of the SIRT3 protein inhibits AMPK phosphorylation and enhances mTOR phosphorylation. As a key metabolic and energy sensor, AMPK mediates a variety of pathological stresses, including metabolic stress and oxidative stress [34]. Overexpression of SIRT3 promoted the phosphorylation of LKB1, which activated the AMPK/mTOR signaling pathway and upregulated autophagy [35]. Interestingly, SIRT3 is also a negative regulator of autophagy, and the overexpression of SIRT3 inhibits the activation of AMPK. Overexpression of SIRT3 leads to deacetylation and activation of manganese superoxide dismutase, which reduces intracellular superoxide content. Furthermore, the reduction in superoxide inhibits AMPK and activates mTOR, leading to autophagy inhibition [36]. This phenomenon has also been found in neurologic disorders. In cerebral ischemic injury, luteolin overexpressed SIRT3 and enhanced mitochondrial oxygen radical scavenging and SOD activity. It inhibited AMPK and activated mTOR [37], which was similar to our findings. We found that Tet inhibited autophagy, suppressed AMPK, and activated mTOR by upregulating SIRT3 expression. These phenomena can be reversed by 3-TYP.

Conclusion

Collectively, the present study provided an evidence that Tet may exert neuroprotective effects by inhibiting autophagy, this may be associated with SIRT3’s inhibitory effect on the AMPK/mTOR signaling pathway.

Acknowledgements

This work was supported by the Medical Science Research Subjects of Hebei Provincial Health Commission (20191190).

Conflicts of interest

There are no conflicts of interest.