Tongqiao Yizhi granule (通窍益智颗粒) repress the nuclear factor kappa-b/nucleotide oligomerization domain-like receptors 3/caspase-1 pyroptosis pathway in the hippocampus to counter vascular dementia in rats

Abstract

OBJECTIVE:

To explore the mechanism by which Tongqiao Yizhi granule (通窍益智颗粒, TQYZKL) intervenes pyroptosis to treat vascular dementia (VaD) in a rat model.

METHODS:

The rat model of VaD was established by two-vessel occlusion (2VO). The rats were randomly divided into Sham group, Model group, Nimodipine group, TQYZKL (6.2 g·kg^-1·d^-1), TQYZKL (12.4 g·kg^-1·d^-1), TQYZKL (24.8 g·kg^-1·d^-1). The Morris water maze (MWM) test was carried out to test the learning and memory function; Hematoxylin-eosin staining and transmission electron microscopy (TEM) to observe the pathological damage in the hippocampus; Tunel fluorescence staining to detect neuronal pyroptosis in the hippocampus. The expression levels of pyroptosis-related proteins, namely Golgi peripheral membrane protein p65 (P65), nucleotide oligomerization domain-like receptors 3 (NLRP3), caspase-1 and Gasdermin D (GSDMD), were detected using Western blotting and reverse transcription polymerase chain reaction. Moreover, the serum levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were determined through the enzyme-linked immunosorbent assay.

RESULTS:

The study revealed that TQYZKL effectively improved the ability of VaD ratsto learn and memorize, relieved the pathological damage in the hippocampus, restored neuronal morphology, and reduced the expression of pyroptosis-related proteins P65, NLRP3, caspase-1, GSDMD-N, IL-18 and IL-1β (P < 0.05).

CONCLUSION:

TQYZKL inhibits neuronal pyroptosis in the hippocampus of VaD rats by regulating nuclear factor kappa-B/NLRP3/caspase-1 signaling pathway, thus exerting a therapeutic effect on VaD in the rats.

1. INTRODUCTION

Vascular cognitive impairment (VCI) refers to an array of clinical syndromes ranging from mild cognitive impairment to vascular dementia (VaD). Mainly arising from cerebrovascular lesions or brain damage, VCI is widely recognized as the second commonest dementia, just next to Alzheimer’s disease (AD). VCI accounts for 15%-20% of all dementia cases in North America and Europe, and but up to 30% in Asia.¹ In China, about 30.6% of dementia patients aged over 55 years present VCI, and about 25%-41% of stroke survivors aged over 60 years may develop VCI within three months.² VCI impedes daily living and social activities of the sufferer, posing heavy economic and psychological burdens on the family and society.¹ There are no specific treatments for VCI. Most of clinical drugs for AD, such as calcium channel blockers, cholinesterase inhibitors and nimodipine receptor blockers, have a short duration of action, a high price, and an unsatisfied efficacy.³ Therefore, studies should further into the pathogenesis of VCI, aiming to discover new therapeutic targets.

Pyroptosis has shown its tight implication in the inflammatory response in cerebral ischemic diseases. As an intermediate condition between necrosis and apoptosis, pyroptosis initiates with the activation of caspase protein family and the release of inflammatory factors interleukin-1β (IL-1β) and interleukin-18 (IL-18). Several studies have shown that pyroptosis is closely related to VaD.^4,⇓,⇓-7 It mediates inflammatory response in many pathological processes, such as brain white matter damage, blood-brain barrier disruption, synaptic plasticity damage, and demyelination, all contributing to the development of cognitive impairment.⁸

Tongqiao Yizhi granule (通窍益智颗粒, TQYZKL) is a formula created by a TCM research team led by Prof. WANG Mingjie. Based on a theory "the root of all diseases lies in Xuanfu depression", TQYZKL has achieved encouraging efficacy in the clinical treatment of VaD. The therapeutic mechanisms involved in this formula have been found to include inhibiting neuronal apoptosis, strengthening the cholinergic system, and reducing mitochondrial oxidative damage.^9,10 Our recent study has confirmed that TQYZKL can increase the microvessel density and the expression of related angiogenic factors in the hippocampus of VaD rats,¹¹ but it is unclear whether these effects have ties with cell pyroptosis. With eyes fixed on the pyroptosis pathway of nuclear factor kappa-B (NF-κB)/nucleotide oligomerization domain-like receptors 3 (NLRP3)/caspase-1, we sought to explore the mechanism underlying TQYZKL in the treatment of VaD using a rat model.

2. MATERIALS AND METHODS

2.1. Reagent and antibodies

Anti-Golgi peripheral membrane protein p65 (P65) antibody (8242, CST, Boston, USA); anti-NLRP3 antibody (AF2155, Beyotime, Shanghai, China); anti-caspase-1 antibody (22915-1-AP, Proteintech, Chicago, IL, USA); Anti-Gasdermin D (GSDMD) antibody (AF4012, Affinity Biosciences, Cincinnati, OH, USA); anti-β-actin antibody (4967S, CST, Boston, MA, USA); horseradish peroxidase-conjugated anti-rabbit secondary antibody (7074S, CST, Boston, MA, USA); reverse transcription kit (R2028, UE, Beijing, China); real-time fluorescence quantitative polymerase chain reaction (PCR) kit (S2024, UE, Beijing, China); Tunel kit (C1090, Beyotime, Shanghai, China); Hematoxylin-eosin (HE) staining kit (C0105S, Beyotime, Shanghai, China); immunohistochemical staining kit (PV-9001, Zhongshan Golden Bridge, Beijing, China); enzyme-linked immunosorbent assay (ELISA) rat Interleukin-18 (IL-18) ELISA kit (ERC010.96, eBioscience, San Diego, CA, USA); ELISA rat Interleukin-1β (IL-1β) ELISA kit (ERC007.96, eBioscience, San Diego, CA, USA).

2.2. Pharmaceutical preparation

The raw herbs of TQYZKL were purchased from the Traditional Chinese Medicine Department of the Affiliated Hospital of Southwest Medical University. Pharmaceutical ingredients have been listed in a previous study.¹² All medicinal materials were prepared into 2 g/mL drug concentrate in the laboratory of the Affiliated Hospital of Southwest Medical University, sterilized and stored in a cool and dry place for backup. Nimodipine (30 mg/tablet) (Hainan Puli Pharmaceutical Co. Ltd., Haikou, China; No.1806003) was prepared into suspension with distilled water before gavage.

2.3. Animals

Eight-week-old male Specific pathogen Free Sprague-Dawley rats (weighing 250-300 g, Liaoning Changsheng Biotechnology Co., Ltd.; No. SCXK [Liaoning] 2020-0001) were fed with a standard diet under standard conditions [(22±2) ℃, 35% ± 5% humidity] in a 12-h light/12-h dark cycle at the Laboratory Animal Research Center of Southwest Medical University. All animal experiments were approved by the Animal Ethics and Welfare Committee of Southwest Medical University of China (No. swum20220132).

2.4. Establishing of a VaD rat model and animal groups

A VaD model was established by permanent occlusion of two-vessel occlusion (2VO).¹³ The common carotid artery was isolated only but not ligated in the sham group. The penicillin (80 000 IU/d) was intramuscularly administered into the rat at 3 d after surgery. The rat memory was evaluated using the Morris water maze (MWM) test at 30 d after surgery. The mean escape latency of the sham group was taken as A, and that of the modeled rats as B. The success of modeling was determined by (B-A)/A × 100% greater than 20%. The rats were subdivided into Sham group, Model group, NMDP group (6.25 mg·kg^-1·d^-1), low-dose TQYZKL group (6.2 g·kg^-1·d^-1), medium-dose TQYZKL group (12.4 g·kg^-1·d^-1) and high-dose TQYZKL (12.4 g·kg^-1·d^-1) randomly (random number table method, n = 6 rats in each group). The Model group and the Sham group were given 3 mL of physiological saline by gavage. Gastric gavage was performed daily in the morning for 30 d. MWM test was performed again after the last gavage to test the learning memory ability of rats.

MWM test: The MWM test was performed according to the method described by Morris.¹⁴ The water maze equipment (R-XM101-Z, Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China) consists of a black circular pool, a platform, and recording system. The pool was divided into four quadrants. A circular, transparent escape platform (10 cm diameter) was placed 2 cm below the surface of the water in the third quadrant of the pool. The rat was given a place navigation test on five consecutive days. Each daily test consisted of four sequential sessions that began with placing the rat in the water facing the wall of the pool. The recording system started to record the time upon placement of the rat in the water. The escape latency (EL) was recorded as the time required for the rat to find the platform. If the rat failed to find the platform within 90 s, it would be guided to the platform by an investigator and was allowed to remain there for 10 s; in this instance, the EL was recorded as 90 s. On the sixth day, the rat was allowed to swim freely in the pool for 60 s without the platform, and the number of crossings through the original platform and the residence time in the third quadrant were recorded.

2.5. Transmission electron microscopy (TEM)

TEM was used to investigate the cellular ultrastructure. Hippocampal tissues were fixed with 2.5% glutaraldehyde and then with 1% osmium tetroxide, subsequently, acetone dehydration, Epoxy resin soaking, and embedding were performed Ultrathin sections (80 nm) were cut using an ultramicrotome with a diamond knife (Leica EM UC7; Leica, Nussolch, Germany), stained with uranyl acetate (10-15 min) and lead citrate (1-2 min), and finnaly observed and photographed (JEM-1400FLASH, JEOL, Tokyo, Japan).

2.6. Hematoxylin and eosin (HE) staining

Hippocampal tissues were formaldehyde-fixed and paraffin-embedded according to established procedures. The paraffin blocks were then cut into 4 mm sections. Having been stained with HE, the sections were examined under magnifications of ×100 and ×200. Images were captured by a light microscope (Leica DM4000B; Leica, Solms, Germany).

2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Staining

The above sections were dewaxed with water routinely and then incubated in Proteinase K for 30 min at 37 °C in the dark. Subsequently, TUNEL action mixture was added, allowed to stand for 60 min at 37 °C in the dark, and then incubated in propidium iodide for 10 min in the dark. Finally, fluorescent images were acquired using a Leica DM5500 B fluorescence microscope connected to a Leica DFC360 FX camera (Leica, Wetzlar, Germany).

2.8. Immunohistochemistry

The sections were dewaxed, dehydrated to transparency. Immunohistochemistry was performed according to instructions. Briefly, the sections were pretreated with sodium citrate buffer at 98 ℃ for 30 min for antigen retrieval, and then the endogenous peroxidase activity was blocked with a peroxidase-blocking reagent for 10 min and then with BSA for 30 min, incubated with the primary GSDMD-N antibody (1∶100) or caspase-1antibody (1∶100) overnight at 4 ℃, and finally with goat anti-mouse biotinylated antibody for 30 min at room temperature. DAB as chromogen was counterstained with hematoxylin.

2.9. Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted from hippocampal tissues at -80 ℃ using Trizol reagent. Primer sequence is shown in supplementary Table 1 (Shanghai Bioengineering Co., Ltd., Shanghai, China).

After reverse transcription, qPCR was performed at predenaturation under 95℃ for 30 s and 40 cycles (denaturation under 95 ℃ for 5 s; annealing under 60 ℃ for 15 s; extension under 72℃ for 25 s). The cycle threshold (CT) value was determined by routine dissolution curve analysis using a fluorescence quantitative PCR instrument (LightCycler® 480Ⅱ; Roche, Switzerland). The relative CT method (△△CT) was used to calculate the relative expression level.

2.10. Western blotting

At －80 ℃, every 0.1 g of hippocampal tissue was added with radio immunoprecipitation assay lysate (including protease inhibitor, phosphatase inhibitor and phenylmet hanesulfonyl fluoride) to extract protein, Bicinchoninic Acid method was used to determine protein concentration. 50 μg of total protein was used for sodium dodecyl sulfate poly acrylamide gel electrophoresis. The membranes were transferred, blocked with 5% skim milk for 1 h, incubated overnight at 4 ℃ with primary antibodies as follows: p65 (1∶1000), NLRP3 (1 ∶ 1000), GSDMD-N (1 ∶ 1000), caspase-1 (1∶1000), β-actin (1∶1000), washed for three times with TBS + Tween, and further incubated with secondary antibody (1∶5000) for 1 h at room temperature. Finally, the protein expression was detected using the enhanced chemiluminescence substrate. Chemiluminescent signals were detected with the Chemi-doc XRS gel documentation system (Bio-Rad, Hercules, CA, USA).

2.11. ELISA

The serum level of IL-18 and IL-1β in the rat was determined through ELISA according to kit instructions. 96-well plates were used and the absorbance wavelength was 450 nm.

2.12. Statistical analysis

The data in a normal distribution were subjected to Kolmogorov-Smirnov test. All values were presented as mean ± standard error of the mean (SEM). SPSS 24.0 (IBM, New York, NJ, USA) was used for data analysis. The EL in the MWM test was analyzed by a two-way repeated measures analysis of variance for the variation across the groups. For other data, the t-test was applied for comparisons between two groups, and one-way analysis of variance for comparisons among multiple groups. A P value < 0.05 was considered as significantly different.

3. RESULTS

3.1. TQYZKL significantly alleviates cognitive impairment in VaD rats

The MWM results showed learning and memory impairment in VaD rats with 2VO. In the navigation tests, the EL of the Model group was significantly longer than that in the Sham group during 2-5 d of training (P < 0.05), but the mean ELs in NMDP and TQYZKL groups were significantly shorter than that in the Model group (P < 0.05) (Figure 1A). At the same time, the number of crossings through the original platform and the residence time in the target quadrant of VaD rats were significantly lower than those in the Sham group in the probe trial. Compared with the rats in the Model group, NMDP and TQYZKL (12.4 and 24.8 g·kg^-1·d^-1) groups had longer dwell time in the original target quadrant, and significantly larger numbers for crossings in the target quadrant (P < 0.05) (Figures 1B, 1C). This indicated that TQYZKL alleviated the cognitive impairment in VaD rats, which is consistent with the previous study in our laboratory.

Figure 1. Morris water maze test.

A: escape latency; B: residence time in the third quadrant; C: the number of platform crossings. Sham and Model group: treated with physiological saline; NMDP group: treated with nimodipine (6.25 mg·kg^-1·d^-1); 6.2, 12.4, 24.8 group: treated with TQYZKL (6.2 g·kg^-1·d^-1), TQYZKL (12.4 g·kg^-1·d^-1), TQYZKL (24.8 g·kg^-1·d^-1) respectively. NMDP: nimodipine; TQYZKL: Tongqiao Yizhi granule. Data are presented as mean ± standard error of the mean (n = 6). Significant differences compared with Sham group were designated as ^aP < 0.05, with Model group as ^bP < 0.05 and with NMDP group as ^cP < 0.05.

3.2. TQYZKL attenuates pathological damage in the hippocampus of VaD rats

HE staining revealed normal morphologies and clear boundaries of hippocampal neurons in the Sham group, while the model group exhibited shrunken neurons with dark-stained nuclei, and even loss of neurons. Surprisingly, NMDP or TQYZKL treatment partially attenuated such neuronal damage and increased the number of neurons in the hippocampus (Figure 2A).

Figure 2. TQYZKL alleviates neuronal pyroptosis in the hippocampus of VaD rats.

A: HE staining (× 400). A1, A7: Sham; A2, A8: Model; A3, A9: NMDP; A4, A10: TQYZKL of 6.2 g·kg^-1·d^-1; A5, A11: TQYZKL of 12.4 g·kg^-1·d^-1; A6, A12: TQYZKL of 24.8 g·kg^-1·d^-1; B: transmission electron microscope result: B1-B6 (× 8000); B7-B12 (× 20 000). B1, B7: Sham; B2, B8: Model; B3, B9: NMDP; B4, B10: TQYZKL of 6.2 g·kg^-1·d^-1; B5, B11: TQYZKL of 12.4 g·kg^-1·d^-1; B6, B12: TQYZKL of 24.8 g·kg^-1·d^-1; C: Tunel staining (× 200): C1, C7, C13: Sham; C2, C8, C14: Model; C3, C9, C15: NMDP; C4, C10, C16: TQYZKL of 6.2 g·kg^-1·d^-1; C5, C11, C17: TQYZKL of 12.4 g·kg^-1·d^-1; C6, C12, C18: TQYZKL of 24.8 g·kg^-1·d^-1; C1-C6: DAPI dyeing; C7-C12: Tunel dyeing; C13-C18: DAPI and tunel dyeing. Sham and Model group: treated with physiological saline; NMDP group: treated with nimodipine (6.25 mg·kg^-1·d^-1); 6.2, 12.4, 24.8 group: treated with TQYZKL (6.2 g·kg^-1·d^-1), TQYZKL (12.4 g·kg^-1·d^-1), TQYZKL (24.8 g·kg^-1·d^-1) respectively. VaD: vascular dementia. HE: hematoxylin-eosin; Tunel: terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI: 4',6-Diamidino-2-phenylindole; NMDP: nimodipine; TQYZKL: Tongqiao Yizhi granule.

TEM showed that the hippocampal neurons in the Sham group had a normal morphology, an intact membrane, and no abnormal organelles and cell contents; but those in the VaD rats were swollen and broken, and some contained pores with release of contents, heavier heterochromatin fixation, and fewer organelles. These changes were attenuated in the drug groups, more obvious in NMDP and TQYZKL (24.8 g·kg^-1·d^-1) groups, as evidenced by reduced cell swelling, release of contents, and chromatin fixation, as well as relatively intact cell membranes (Figure 2B).

3.3. TQYZKL alleviates neuronal pyroptosis in the hippocampus of VaD rats

TUNEL staining could display the broken DNA fragments in apoptotic or pyroptotic cells. The model group exhibited a large number of positive neurons in the hippocampus. After TQYZKL intervention, the number of positive neurons in the hippocampus decreased significantly (Figure 2C).

3.4. TQYZKL ameliorates VaD through repressing the NF-κB/NLRP3/caspase-1 pathway

To determine whether the neuroprotective mechanism of TQYZKL is related to the inhibition on the NF-κB/ NLRP3/caspase-1 pathway, we detected the effects of TQYZKL on the expression levels of key proteins, including P65, NLRP3, caspase-1, GSDMD-N by Western blotting and qPCR. The mRNA levels and protein levels of P65, NLRP3, caspase-1, GSDMD-N in the VaD rats were significantly increased, which were then strongly suppressed by TQYZKL (12.4 and 24.8 g·kg^-1·d^-1) (Figures 3A-3C).

Figure 3. TQYZKL ameliorates VaD through repressing the NF-κB/NLRP3/caspase-1 pathway by q-PCR and western blotting.

A: mRNA levels of P65, NLRP3, caspase-1, GSDMD. A1: P65; A2: NLRP3; A3: caspase-1; A4: GSDMD; B: protein levels of P65, NLRP3, caspase-1, GSDMD-N. B1: P65; B2: protein levels of NLRP3; B3: caspase-1; B4: GSDMD; C: representative Western blot. Sham and Model group: treated with physiological saline; NMDP group: treated with nimodipine (6.25 mg·kg^-1·d^-1); 6.2, 12.4, 24.8 group: treated with TQYZKL (6.2 g·kg^-1·d^-1), TQYZKL (12.4 g·kg^-1·d^-1), TQYZKL (24.8 g·kg^-1·d^-1) respectively. TQYZKL: Tongqiao Yizhi granule; NF-κB: Nuclear factor kappa-B; NLRP3: nucleotide oligomerization domain-like receptors 3; q-PCR: quantitative real-time polymerase chain reaction; P65: GRASP65 protein; GSDMD: Gasdermin D; NMDP: nimodipine. Data are presented as mean ± standard error of the mean (n = 4). Significant differences compared with Sham group were designated as ^aP < 0.05, with Model group as ^bP < 0.05 and with NMDP group with ^cP < 0.05.

Expression levels of caspase-1 (Figure 4A) and GSDMD (Figure 4B) in the hippocampus of rats were measured by IHC. Relative to the Sham group, their higher expression levels were detected in the Model group. Relative to the Model group, significant reductions were found in NMDP and TQYZKL groups (12.4 and 24.8 g·kg^-1·d^-1).

Figure 4. TQYZKL ameliorates VaD through repressing the NF-κB/NLRP3/caspase-1 pathway by IHC and ELISA.

A: expression of caspase-1 protein in rat hippocampus (× 200); A1: Sham; A2: Model; A3: NMDP; A4: TQYZKL of 6.2 g·kg^-1·d^-1; A5: TQYZKL of 12.4 g·kg^-1·d^-1; A6: TQYZKL of 24.8 g·kg^-1·d^-1; B: expression of GSDMD protein in rat hippocampus (× 200), B1: Sham; B2: Model; B3: NMDP; B4: TQYZKL of 6.2 g·kg^-1·d^-1; B5: TQYZKL of 12.4 g·kg^-1·d^-1; B6: TQYZKL of 24.8 g·kg^-1·d^-1; Dyeing method of all pictures are the immunohistochemical method; C: expression of IL-18 and IL-1β in serum of rats. Sham and Model group: treated with physiological saline; NMDP group: treated with nimodipine (6.25 mg·kg^-1·d^-1); 6.2, 12.4, 24.8 group: treated with TQYZKL (6.2 g·kg^-1·d^-1), TQYZKL (12.4 g·kg^-1·d^-1), TQYZKL (24.8 g·kg^-1·d^-1) respectively. TQYZKL: Tongqiao Yizhi granule; NF-κB: nuclear factor kappa-B; NLRP3: nucleotide oligomerization domain-like receptors 3; IHC: immunohistochemistry; ELISA: enzyme-linked immunosorbent assay; IL-18: interleukin-18; IL-1β: interleukin-1β; NMDP: nimodipine. Data are presented as mean ± standard error of the mean (n = 4). Significant differences compared with Sham group were designated as ^aP < 0.05, with Model group as ^bP < 0.05 and with NMDP group with ^cP < 0.05.

In addition, we detected the serum levels of IL-18 and IL-1β in rats by ELISA. Compared with those in the Sham group, the serum levels of IL-18 and IL-1β increased significantly in the Model group, but these alterations were significantly reversed by TQYZKL (Figure 4C).

4. DISCUSSION

Studies have indicated that neuroinflammation is a key factor in dementia after cerebrovascular disease.¹⁵ Pyroptosis is recognized as a potent trigger of neuroinflammation during cerebral ischemic injury, and inhibition on pyroptosis may alleviate ischemic brain injury.^6,16 Consequently, pyroptosis is becoming a potential therapeutic target to treat neuroinflammation and VaD. In the present study based on the rat model, we for the first time verified that TQYZ counters VaD through repressing the pyroptosis-assosiated NF-κB/ NLRP3/caspase-1 pathway.

Pyroptosis is a programmed cell death discovered in recent years. During pyroptosis, caspase-1 is mainly mediated by inflammatory vesicles with their own pattern recognition receptors (PRRs) (e.g., NLRP1, NLRP3, AIM2). Apoptosis-related spot protein ASC is recruited to pro-caspase-1 and activate pro-caspase-1 to caspase-1. As a consequence, the production of inflammatory factors IL-18 and IL-1β is induced, and meanwhile inflammatory vesicles are activated to specifically cleave the GSDMD protein into an active N-terminal fragment (GSDMD-N), which can bind tightly to the plasma membrane and produce pores with a diameter of 10-20 nm, causing permeable swelling of cells. As the cells rupture, cytosolic contents are released, and a cascade of inflammatory responses are evoked.¹⁷

NLRP3, as an initiator of aseptic inflammatory response of the central nervous system after cerebral disease, is essential for the activation of caspase-1 and the secretion of IL-1β and IL-18 in response to bacterial and endogenous stimuli.^18,19 With the activation of NF-κB, the expression of NLRP3 is up-regulated, making it the first signal of inflammatory response.²⁰ GSDMD is a protein closely related to pyroptosis, and a most important substrate of caspases. GSDMD has two conserved structural domains: N-terminal effector domain and C-terminal repressor domain; N-terminal is the main functional domain, which acts to inhibit proliferation and arouse pyroptosis, while C-terminal has autoinhibitory and protective functions. However, once their functional balance is disrupted (such as release of C-terminus due to mutation, translational modification and structural damage), the effect of N-terminal will be magnified; then, with the release of a large amount of inflammatory factors, including IL-1β and IL-18, inflammation and pyroptosis are induce.²¹

As a TCM formula based on the theory of Xuanfu, TQYZKL is composed of "wind-expelling medicine + tonics + insects". Wind-expelling medicine Gegen (Radix Puerariae Lobatae) is the monarch drug which can penetrate into the body through the skin pores to expel wind. Renshen (Radix Ginseng), Huangqi (Radix Astragali Mongolici) and Lingzhi (Ganoderma Lucidum) are ministerial medicines to tonify deficiency, rejuvenate spirit, and transport Shenji. Insects Shuizhi (Hirudo) and Dilong (Pheretima Aspergillum) are adjuvant drugs to dissipate phlegm and blood stasis. These three types of drugs work together to tonify deficiency, revitalize spirit, as well as remove the phlegm. Various natural medicines in TQYZKL can strongly dredge brain collaterals by invigorating Qi, removing blood stasis, expelling wind, as well as promoting the circulation of Qi by aromatic herbs. Studies have found that TQYZKL is especially suitable for the prevention and treatment of cerebro-vascular diseases. Our previous research indicates that TQYZKL can protect neurons via quelling neuro-inflammation in VaD rats after cerebral ischemia.

The pathological mechanisms underlying VaD are complicated. It has been shown that the NLRP3/caspase-1/GSDMD pathway, a pathway related with pyroptosis, is activated in VaD.²² Pyroptosis causes neuronal loss, leading to neurological deficits and functional impairment of the brain; meanwhile, intracellular inflammatory factors IL-18 and IL-1β are continuously released to activate microglia and astrocytes, thus causing infiltration of peripheral immune cells into the brain. Therefore, with the aggravation of the inflammatory response, brain function is further damaged, forming a vicious cycle. In this study, we first observed the pathological damage of the hippocampus in VaD rats. TEM showed that the hippocampal neurons in VaD rats underwent obvious pyroptosis, which was then effectively curbed by TQYZKL. Subsequently, we observed hippocampal pyroptosis by TUNEL staining. However, this method could not distinguish apoptosis from pyroptosis. So, we further detected the expression of pyroptosis-related proteins in VaD rats. All were markedly upregulated, including P65, NLRP3, GSDMD-N, caspase-1. In addition, the serum levels of proinflammatory mediators, such as IL-1β and IL-18, increased significantly. But these increases were all repressed after TQYZKL treatment. Our results showed that TQYZKL could inhibit pyroptosis and related inflammatory responses through inactivating the NF-κB/ NLRP3/caspase-1 pathway in VaD rats.

In conclusion, our study revealed that TQYZKL inhibits neuronal pyroptosis of hippocampal neurons of vascular dementia rats by repressing the NF-κB/NLRP3/caspase-1 signaling pathway, thus exerting a therapeutic effect.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.