Salvianolic acid A improves chronic cerebral ischemia through TLR4/MAPKs/AP-1 signaling pathway

doi:10.21203/rs.3.rs-1635580/v1

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Salvianolic acid A improves chronic cerebral ischemia through TLR4/MAPKs/AP-1 signaling pathway

https://doi.org/10.21203/rs.3.rs-1635580/v1

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Chronic cerebral hypoperfusion (CCH) is a clinical syndrome. Long-term chronic cerebral low perfusion will lead to insufficient blood supply to the brain, this leads to a pathological cascade. Salvianolic acid A (SAA) is a water-soluble phenolic acid contained in Salvia miltiorrhiza Bge., which has regulated effect on cardiovascular and cerebrovascular diseases. We established a chronic bilateral common carotid artery occlusion (BCCAO) model to investigate the improvement of SAA on cognitive dysfunction after chronic cerebral ischemia, and further explore whether it has a protective effect on hippocampal neuron damage. The mRNA changes of BCCAO model rats before and after SAA administration were analyzed based on RNA-Seq technique. Combined with GO and KEGG analysis results, PPI network analysis was performed for the major differential genes. Finally, the results were verified by western blot and enzyme-linked immunosorbent assay. After SAA treatment in BCCAO rats, behavioral experiments showed that SAA could improve learning and memory dysfunction caused by BCCAO model in rats. Moreover, it can significantly improve the hippocampal damage caused by long-term cerebral ischemia. Differential gene analysis revealed that there were multiple gene and pathway changes before and after SAA treatment. The genes we screened included TLR4, MyD88, Fos, Jun, ERK1/2, JNK and P38. Western blotting and enzyme-linked immunosorbent assay showed that SAA could regulate the TLR4/MAPKS/AP-1 signaling pathway, reduce the release of inflammatory factors, improve the nerve injury caused by brain ischemia in rats, and playing neuroprotective effects.

Chronic cerebral hypoperfusion

Salvia miltiorrhiza Bge.

salvianolic acid A

neuroprotective

Chronic cerebral hypoperfusion is a clinical syndrome, it is not easy to detect after the illness, leading to high death rates [1]. Ischemic brain injury accounts for about 85% of the incidence of stroke [2], and is one of the three major diseases causing human death. Chronic cerebral ischemia is mainly caused by long-term chronic cerebral hypoperfusion, which refers to insufficient blood flow, defective perfusion or dysfunction of automatic regulation. Chronic refers to a continuous state or a swing state that is characterized by a repetitive phase of inefficient functional congestion. This phenomenon can affect the entire brain or specific regions, leading to cerebral infarction, vascular cognitive dysfunction, etc. Chronic cerebral ischemia is closely related to neuronal injury and cognitive ability [3], and the previous research focuses on the study of chronic cerebral ischemic mechanism [4–6]. Ischemic cascade reaction occurs after cerebral ischemia, from energy supply depletion to membrane depolarization, excitatory amino acid release, ion inflow, free radical production, blood-brain barrier destruction, edema and inflammation, brain damage will eventually be aggravated [7]. The process after cerebral ischemia involves multi-channel, multi-target, including but not limited to immune inflammation, calcium signaling pathways, neuroactive receptor and ligand interactions, apoptosis, and so on.

Salvia miltiorrhiza Bge. is bitter in taste and cold-natured. It has the effects of promoting blood circulation and removing blood stasis, clearing heat from the blood and resolving swelling, tranquilizing the mind. The plant name was confirmed at http://www.theplantlist.org/. In the compatibility of traditional Chinese medicine (TCM), Salvia miltiorrhiza Bge. can improve ischemic stroke [8, 9] and reduce cerebral heart syndrome caused by stroke [10]. Salvia miltiorrhiza Bge. extract attenuates permanent cerebral ischemia [11] and can anxiolytic [12]. Salvia miltiorrhiza Bge. has a variety of medicinal effects while ensuring good safety. Studies have shown that Salvia miltiorrhiza Bge. can inhibit thrombosis by improving blood circulation [13]. Salvia miltiorrhiza Bge. can also be used to treat coronary heart disease [14], cerebrovascular diseases [15], Alzheimer's disease [16], Parkinson's disease [17], cirrhosis [18, 19], and other diseases. Diterpenoid ketones and hydrophilic phenolic acids in Salvia miltiorrhiza Bge. are the main bioactive components [20].

High performance liquid chromatography (HPLC) found that SAA is an important water-soluble phenolic compound in Salvia miltiorrhiza Bge.. Du, et al found that SAA had a good protective effect on myocardial ischemia [21]. SAA also has a good effect in cerebrovascular diseases, and can alleviate learning and memory dysfunction in mice caused by cerebral ischemia and reperfusion [22]. SAA can improve ischemic stroke in vivo ischemia and in vitro oxygen and sugar deprivation models [23]. There are many studies on the mechanism of SAA in apoptosis, immune inflammation [6], calcium overload, oxidative stress and other aspects, but the main target and mechanism of its role are not clear, and whether SAA plays a role through multiple pathways is still unknown. Therefore, we conducted PPI network analysis, KEGG analysis and GO analysis based on the changes of major differential genes in rats before and after SAA treatment to investigate the main mechanism of SAA in the treatment of chronic cerebral ischemia.

Materials and reagents

SAA was isolated from Salvia miltiorrhiza Bge. by the group of Dr. Jinhui Wang (Harbin Medical University), as a lyophilized powder with purity 98.5%. The structure of salvianolic acid A was confirmed by ¹H NMR (600 MHz, DMSO-d6). GAPDH, Gel preparation kit, horseradish enzyme labeled goat anti-mouse/rabbit IgG, β-actin, RIPA lysate, PMSF, 5 × Loading Buffer (Beyotime Biotechnology Co., Ltd-China); TLR4 antibody (abcam-U.K); MyD88 antibody, p44/42 MAPK antibody, p38 MAPK antibody, Phospho-p38 MAPK antibody, SAPK/JNK antibody, PhosphoSAPK/JNK antibody, c-Jun antibody, Phospho-c-Jun antibody, c-Fos antibody, Phospho-c-Fos antibody (affinity-USA); Phosphop44/42 antibody (CST-USA); PageRuler™ Prestained Protein Ladder (Thermofisher-USA); Rat IL-1β, IL-6, IL-12, enzyme-linked immunosorbent assay kit (Elabscience-China).

Animals

SD rats weighed 250 ± 20 g. Purchased from Liaoning Changsheng Biotechnology Co., LTD. License Number: SYXK (Liao) 2020-0001, experimental animal quality certificate number: 211002300062500. Animals can drink and eat freely. The experiment is carried out one week after rats adapt to the environment. The room temperature is set to 22 ± 2 °C. All experimental operations were following the regulations of the Animal Ethics Committee of Shenyang Pharmaceutical University, China.

Chronic bilateral common carotid artery occlusion (BCCAO) model

SD rats were randomly divided into control group, sham group, BCCAO group, SAA (1.5 mg/kg) group, SAA (3.0 mg/kg) group, SAA (6.0 mg/kg) group and edaravone (5.0 mg/kg) group [24].The BCCAO model established by Sarti et al [25],rats were anesthetized by inhalation of isoflurane. Then rats' right common carotid artery was ligated at both ends, and vessels were cut off from the middle. One week later, ligated the left common carotid artery. In the sham group, bilateral common carotid arteries were separated but not ligated.

Behavioral tests

1) Determination of rat autonomous activity

The top of the autonomous activity box with a cassette camera and an infrared light source. The rats were placed in the activity box, and the video was recorded. After the rats explored freely for 5 min, the video was stopped. EthoVision XT 8.0 (Noldus Information Technology, Netherlands) was used to analyze the movement distance and speed of the experimental animals.

2) New object recognition

The internal size of the rat autonomous activity box was 50 cm×50 cm×60 cm (length × width × height). The experiment was divided into three stages. Adaptive stage: the rats explored in the box without any object for 5 min; Training stage: Two identical objects were placed symmetrically in the box, and the rats were free to explore for 5 min and videotaped; Test stage: one of the objects was replaced with a new object, and the other was unchanged, the rats explored for 5 min and videotapedv [26]. EthoVision XT 8.0 was used to analyze. Recognition index (RI) = TA2/(TA1+TA2) and Discrimination index (DI) = (TA2-TA1)/(TA1+TA2) were calculated.

3) Y maze

The Y maze has three arms A, B, and C (each arm has an angle of 120°), each arm is 50cm×18cm×35cm (length × width × height), and their intersection is the isolation zone (zone O). One arm was set as the initial arm, and rats were placed in the arm. The entry of rats into the arm within 8 min was recorded, and the percentage of continuous entry into three different arms was calculated. Spontaneous alternation reaction rate = Total number of consecutive entry into 3 different arms (n) / [Total number (N) -2].

4) Morris water maze

Morris water maze is a black pool with a diameter of 200 cm and a height of 60 cm. It has four quadrants, and the water temperature is 22 ± 2 °C. A digital camera hangs directly above the maze to record each rat's swim. The platform was set in the first quadrant, and the rats were placed facing the bucket in quadrant 2 and quadrant 4 successively. Recorded the movement trajectory of rats, and the time to find the platform within 90 s as the escape latency. When more than 90 s, the escape latency was 90 s and the rats were guided to the platform for 20 s [27]. The score on the day was the mean of the two escape latency periods. On the sixth day, the platform was removed, and the rats were placed from the same water entry point in the first quadrant, and the number of times they crossed the original platform within 90 s was measured. EthoVision XT 8.0 software was to record the daily training of rats.

HE staining

After perfusion, the whole brain was taken out and fixed in 4% formalin solution, placed in 4 ℃ refrigerator for about 8 hours, and then dehydrated and embedded. Place paraffin sections (5 μm) in xylene I for 10 min, xylene II for 10 min, absolute ethanol I for 3 min, absolute ethanol II for 3 min, 95% ethanol I for 3 min, 95% ethanol II for 3 min, 80% ethanol for 1 min, deionized water for 2 min, then stained with hematoxylin solution for 5 min, wash away the unconjugated hematoxylin solution, differentiate with 1% hydrochloric acid ethanol solution for 30 s, use 1% ammonia to return to blue (5 s), wash with water (2 min), and stain with 0.5% eosin solution for 3 min, wash with distilled water (2 s), 80% ethanol for 2s, 95% ethanol for 3min, anhydrous ethanol for 5min, anhydrous ethanol for 5min, carbolic acid xylene for 5min, xylene for 5min, xylene for 5min, carbolic acid xylene for 5min, xylene for 5min, xylene for 5min, xylene for 5min, xylene for 5min and xylene for 5min. Seal with neutral gum.

The variation of differential gene expression was detected by the RNA-Seq technique

The hippocampal tissues of 3 rats in each group were collected from the sham group, model group and SAA group (6.0 mg/kg). Place it in the RNA hold and fully infiltrate it. Send the samples to BGI for testing. The differences of mRNA expression in hippocampus after treatment were statistically analyzed.

Western Blotting

Lytic the hippocampus tissue, centrifuge at 12000 rpm (10 min) and take the supernatant for experiment. BCA method was used to determine the protein concentration. The voltage in the concentrated gel is 60 V, then adjusted to 110 V. The PVDF membrane was activated in methanol and covered with gel. Then, the electroporate is carried out with a constant current. According to the different molecular weight of protein, the electrokinetic time is different. The PVDF membrane was sealed with 5% skimmed milk powder for 2-3 h to eliminate the background. The membrane was hybridized with primary antibody. Incubate a room temperature for 30 min and then incubate overnight at 4 °C. After, the membrane was washed 3 times with TBST. Incubate the membrane in the secondary antibody for 1 h. Then the membrane was washed with TBST (3 times). At last, add an appropriate amount of ECL luminescent liquid, and exposure to the machine.

ELISA

Tissue was added to PBS and homogenized. Centrifuge for 10 min at 2-8 °C, and take the supernatant for testing. The plate was incubated at 37 °C for 90 min. Add 100 μl of biotinylated antibody working solution, and incubate at 37 °C for 1 hour. Pour out the liquid, add washing water for 1 min, pat dry with absorbent paper, and repeat 3 times. Add 100 μl of enzyme conjugate working solution, and incubate at 37 °C for 30 min. Wash the plate 5 times, add 90 μl of substrate solution (TMB) to each well, and incubate at 37 °C for 15 min. Finallt, add 50 μl stop solution to stop the reaction. Measure the optical density with a wavelength of 450 nm.

Statistical analysis

The data were collected by SPSS 21.0 software and R language; Graphpad prism 7 was used for image processing. One way analysis of variance (one-way ANOVA) is used for data statistics. When the variance is homogeneous, LSD is used for test. When the variance is uneven, Dunnett't3 is used for analyze. The data are expressed as mean ± standard error (mean ± SEM). p< 0.05 was considered as significant difference.

HPLC and ¹H-NMR of SAA

SAA as a lyophilized powder with purity 98.5% (Fig. 1A). The structure of SAA was confirmed by ¹H NMR (600 MHz, DMSO-d6) (Fig. 1B).

Effects of SAA on cognitive dysfunction and hippocampal pathological changes in rats

SAA is a water-soluble phenolic acid compound (Fig. 2A). Rats were treated with SAA three weeks after BCCAO model, and behavioral detection was performed in the fourth week (Fig. 2B).

According to the analysis of rats' movement speed and distance (Fig. 2C, D), there were no significant changes after the BCCAO model and SAA treatment, indicating that BCCAO model and SAA injection has no effect on the autonomous activity of the rats. In the new object recognition test (Fig. 2E, F), the recognition index and discrimination index of the BCCAO model rats were significantly decreased, while the SAA medium and high dose groups and edaravone groups were up-regulated. Suggests that SAA can improve the recognition and memory of rats. Y maze was used to investigate the spatial learning and memory ability of rats (Fig. 2G, H). There was no significant difference in the total number of channel entries among the groups, suggesting that BCCAO model and SAA did not affect the motor function of rats. The spontaneous alternation response rate of BCCAO rats was significantly decreased, indicating that the rats had spatial learning and memory impairment. Compared with the model group, the spontaneous alternation reaction rate of the SAA and edaravone groups was significantly increased, indicating that the drug had a protective effect on the BCCAO model rats and could improve the learning and memory function of the rats.

HE staining results were shown in Fig. 2I, the hippocampal neurons of the control group were dense and orderly arranged, with abundant cytoplasm, complete cell membrane and nuclear membrane, and clear nucleoli. The neurons were arranged regularly, and the cell membrane and nuclear membrane were intact in the sham group. The hippocampal neurons in the BCCAO group showed obvious phenomena such as loose and irregular arrangement, swelling and vacuolar degeneration, decreased cell number, and nuclear shrinkage and dissolution. After SAA treatment, the hippocampal damage could be improved. The hippocampal neurons in SAA and edaravone groups were regular, and the number of cells recovered.

In the orientation navigation experiment, with the increase of training period, the swimming distance of each group will decrease. In addition, the total distance between SAA (6.0 mg/kg) group and edaravone group was significantly reduced compared with the BCCAO group on the 3rd, 4th and 5th days of training. With the change of training days, the escape latency of each group also decreased gradually. The escape latency of BCCAO rats increased significantly on days 3,4, 5 and decreased significantly in the high-dose SAA group and the edaravone group (Fig. 3A, B). After the platform was removed from the first quadrant, the number of crossing the platform and the residence time in the target quadrant of the rats in the model group were significantly reduced (Fig. 3C, D). Compared with the BCCAO group, the frequency of crossing the platform in SAA group and edaravone group was significantly increased. The residence time in the target quadrant of SAA (6.0 mg/kg) group and edaravone group was significantly prolonged. The swimming tracks of the SAA high-dose group and edaravone group were mainly concentrated in the first quadrant, while the swimming tracks of the BCCAO group were distributed in each quadrant (Fig. 3E). There was no significant change in the swimming speed of each group within 6 days (Fig. 3F), suggesting that SAA had no significant effect on the exercise ability of rats. In conclusion, we found that SAA can improve the spatial learning and memory ability of BCCAO rats.

Changes of differential genes in rats before and after SAA treatment

The total differential genes before and after treatment were analyzed by R language. Then, the volcano map was obtained (Fig. 4). 205 genes were significantly changed in the sham group and the model group. Statistics showed that 77 genes were down-regulated and 128 genes were up-regulated. There were 220 gene changes between the BCCAO group and SAA (6.0 mg/kg) group, of which 74 genes were down-regulated and 146 genes were up-regulated.

According to the BGI gene data, we found that many genes have changed before SAA treatment. The two online websites of KOBAS and DAVID are used to analyze these differential genes and get the statistical data of the KEGG signal pathway. The results of KEGG analysis of these differential genes suggest that multiple pathways have changed, including MAPKs signaling pathway, Toll-like receptor signaling pathway, calcium signaling pathway, neuroactive ligand-receptor interaction, etc. (Fig. 5A). The GO analysis of the online website (divided into three parts: cellular component, CC; molecular function, MF; biological process, BP) results suggests the biological process of the immune system has major changes before and after SAA treatment, mainly involving the regulation of glial cells, T cells and other immune cells (Fig. 5B, C). We focused on the major changes in the above four pathways and screened related genes for PPI network analysis (Fig. 5D). The degree of association of each gene from the inside to the outside is from large to small, genes in the middle having the highest degree of association and the darkest color. From the PPI network, we found that Fos, Jun, TLR4, etc. are closely related to the Toll-like receptor signaling pathway. Therefore, we researched TLR4, MyD88, Fos, Jun, Erk1/2, JNK, P38, and other related genes, and analyzed the changes in the protein levels of related genes.

Effect of SAA on expression of related proteins after chronic cerebral ischemia

After differential gene analysis, it is found that the main genes that change before and after SAA administration are TLR4, MyD88, Fos, Jun, Erk1/2, JNK, P38, etc., which are closely related to the Toll-like receptor signaling pathway and the MAPKs pathway. Therefore, we analyzed the expression of the above proteins. As shown in Figs. 6A, B, TLR4 protein expression was significantly up-regulated after the BCCAO model, and the expression of TLR4 was decreased in a dose-dependent manner after SAA treatment (Fig. 6B). High doses and positive drugs were significant. The expression of MyD88 increased after modeling, but there was no significant difference (Fig. 6B). The effect of SAA on MAPKs signaling pathway was shown in Fig. 6C, D. The protein expression levels of Erk1/2, JNK, and P38 showed no significant changes in the model and after administration. However, the expression levels of P-JNK and P-P38 proteins were significantly increased after modeling, and SAA decreased the phosphorylation levels of JNK and P38 proteins in a dose-dependent manner, which was significant in the middle and high dose groups. The phosphorylation level of Erk1/2 showed no significant change after modeling. The expression of p-c-Fos and p-c-Jun protein in BCCAO model rats is significantly up-regulated (Fig. 6E, F). The SAA middle and high dose groups can significantly reduce the expression of the p-c-Fos protein. SAA significantly reduced the expression of p-c-Jun in a dose-dependent manner.

Effect of SAA on expression of IL-1β, IL-12 and IL-6 in hippocampus

Investigating the regulatory effect of SAA on the expression of inflammatory factors in the hippocampus of rats (Fig. 7). IL-1β and IL-6 were significantly up-regulated after modeling, and the expression of IL-1β and IL-6 was decreased after SAA and positive drug treatment. These results indicate that SAA can reduce the expression of inflammatory factors in the hippocampus.

In this study, SAA was found to regulate Toll-like receptor signaling, which in turn regulates the expression of inflammatory factors. In the pre-exploration, our team found that the BCCAO model could cause chronic cerebral ischemia after four weeks. After chronic cerebral ischemia, the ischemic sensitive areas such as hippocampus are greatly affected. Pathological sections showed vacuolar degeneration and loose arrangement of cells in the hippocampus. At the same time, learning and cognitive functions are closely related to the hippocampus, and long-term chronic cerebral ischemia will eventually lead to learning and memory dysfunction in rats [28–30]. The behavioral test showed that chronic cerebral ischemia could damage the cognitive function of rats. However, SAA can significantly improve the cognitive dysfunction of rats, and there is similar or better than edaravone. Edaravone, a free radical scavenger, processes strong biological activities including antioxidant, neuroprotective and anti-inflammatory effects [31]. It is commonly used to treat cerebral ischemia. SAA showed similar improvement to edaravone and was superior to edaravone in some tests. These tests prove that SAA plays an important role in improving brain injury and provide a basis for further exploring the mechanism.

Our team further investigated the protective mechanism of SAA on hippocampus injury. KEGG pathway enrichment analysis showed that MAPKs signaling pathway, Toll-like receptor signaling pathway, calcium signaling pathway, and neuroactive ligand-receptor interaction all changed before and after treatment. We performed a PPI network analysis. The closely interacting genes are mainly: TLR4, MyD88, Fos, Jun, Erk1/2, JNK, P38, etc. These genes are closely related to the Toll-like receptor signaling pathway and MAPKs pathway. At the same time, according to GO enrichment analysis, it is found that the main changes in the biological process are T cell-mediated immunity and the regulation of T cell-mediated immunity. It is known that chronic cerebral ischemia is usually accompanied by chronic neuroinflammation, aggravating cerebral ischemia injury, which is also an important pathogenic mechanism of cognitive decline [32, 33].

Studies have found that TLR4 can regulate Th1 and Th2 immune responses through downstream mechanisms [34]. The MAPKs pathway can be activated by a variety of factors to regulate the secretion of inflammatory cytokines related to T cell-mediated immune response. The results showed that TLR4 expression increased after cerebral ischemia, but TLR4 activation did not promote the activation of downstream MyD88 protein, indicating that SAA may not regulate cerebral ischemia through MyD88-dependent pathways. One study showed that protein abundance in cells is mainly controlled by the level of translation, and proteins are different from mRNA in terms of half-life, synthesis rate, and quantity [35]. There are also reports that after stimulating TLR in MyD88 knockout mice, JNK and NF-κB are activated [36]. This may explain the difference in mRNA and protein expression of MyD88. It shows that TLR4 can also regulate the MAPKs signaling pathway without the MyD88 pathway. Subsequently, we investigated the phosphorylation levels of Erk1/2, JNK, and P38 proteins, and we found that the phosphorylation levels of JNK and P38 proteins in the hippocampus of model rats significantly increased, while the phosphorylation level of Erk1/2 proteins did not change significantly. Toll-like receptor signaling pathway will eventually activate the expression of transcription factor AP-1. AP-1 is an inducible transcription factor that participates in gene transcription regulation after binding to specific sequences in the promoter region of target genes. The DNA binding activity of AP-1 increased after ischemia, suggesting that AP-1 is involved in the neuroprotective effect induced by ischemia [37]. SAA treatment can reduce the phosphorylation level of AP-1 protein.

AP-1 is closely related to inflammation and tissue damage, and dysregulation of AP-1 activity can cause inflammation. Besides, inflammation of endothelial cells during cerebral ischemia will activate macrophages and oligodendrocytes release pro-inflammatory factors. With continued over-activation of microglia, inflammatory factors are produced that cause neuroinflammatory damage [38]. The inflammatory response can aggravate the damage to the nervous system. Therefore, inhibiting the inflammatory response after ischemia, interfering with the inflammatory process, and reducing the damage to the nervous system is one of the methods to treat the neuronal damage after ischemic brain injury and improve the ability of learning and memory. SAA can reduce pro-inflammatory factors such as IL-6 and IL-1β, and improve neuroinflammation after chronic cerebral ischemia. IL-6 induces the differentiation and production of antibodies in B cells and the proliferation and differentiation of T cells participate in immune response, and acts as a trigger for inflammatory reactions. IL-1β participates in the regulation of immune response and cell proliferation and differentiation and mediates signal transduction [39]. From the results, the effect of SAA was more significant than edaravone in reducing pro-inflammatory factors.

In general, SAA reduces pro-inflammatory factors and regulates Toll-like receptor signaling pathway better than edaravone. It plays an important role in improving rat cognitive dysfunction and reducing inflammatory factors. SAA may improve hippocampal damage in rats by regulating the TLR4/MAPKs/AP-1 signaling pathway.

Ethics approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures were carried out in accordance with the Animal Ethics Committee, Shenyang Pharmaceutical University, China (SCXK (Liao) 2020-0001).

Consent to participate

Not applicable.

Consent for publication

All authors reviewed and agreed for the publication of this manuscript.

Availability of data and materials

Data and materials will be provided by corresponding authors upon proper request.

Competing interests

All authors declare no conflicts of interest.

Funding

This study was supported by Scientific Research Foundation of the Education Department of Liaoning Province (LJKZ09562021), Scientific Research Staring Foundation for the Returned Overseas Scholars, Shenyang Pharmaceutical University (No: GGJJ2021101), and National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2018ZX09735005).

Author Contribution

Chunli Li designed research; Fei Bai, Sheng Yin, Yue Xu, Jian Wang conducted in vivo and in vitro pharmacodynamics experiments. Qingqing Hu, Renkuan Sun, Jinhui Wang, Baofeng Yang provided salvianolic acid A and edaravone, and provided the structure of salvianolic acid A. Chunli Li and Fei Bai wrote the paper. All authors have approved the final version of the manuscript. The authors declare no conﬂict of interest.

Acknowledgements

Choi BR et al (2016) Characterization of White Matter Injury in a Rat Model of Chronic Cerebral Hypoperfusion. Stroke 47:542–547
Martha SR et al (2019) Acid-Base and Electrolyte Changes Drive Early Pathology in Ischemic Stroke. Neuromol Med 21:540–545
Cechetti F et al (2012) Forced treadmill exercise prevents oxidative stress and memory deficits following chronic cerebral hypoperfusion in the rat. Neurobiol Learn Mem 97:90–96
Antipenko EA et al (2016) The system stress-limiting action of mexidol in chronic cerebral ischemia. Zh Nevrol Psikhiatr Im S S Korsakova 116:28–31
Edrissi H et al (2016) Thompson CS. Microparticles generated during chronic cerebral ischemia increase the permeability of microvascular endothelial barriers in vitro. Brain Res 1634:83–93
Ling Y et al (2021) Salvianolic acid A alleviated inflammatory response mediated by microglia through inhibiting the activation of TLR2/4 in acute cerebral ischemia-reperfusion. Phytomedicine 87:153569
Wang YL et al (2011) Status and future of neuroprotective agents in the treatment of stroke. Chin J New Drugs 20:985–987
Zhou L et al (2005) Danshen: an Overview of its Chemistry, Pharmacology, Pharmacokinetics, and Clinical Use.J. Clin Pharmacol 45:1345–1359
Huang K et al (2015) Expression of TLR2 and TLR4 in children with henoch Schonlein Purpura and their relationship with Th1 and Th2 immune responses. J Hainan Med Coll 21:1664–1667
Long Y et al (2021) Research progress of traditional Chinese medicine in the treatment of multiple organ injury caused by ischemic stroke. Chin Traditional Herb Drugs 690:2106–2116
Fei YX et al (2017) Salvia miltiorrhiza Bunge (Danshen) extract attenuates permanent cerebral ischemia through inhibiting platelet activation in rats. J Ethnopharmacol 207:57–66
Lin YS et al (2021) Anxiolytic effect of an extract of Salvia miltiorrhiza Bunge (Danshen) in mice. J Ethnopharmacol 264:113285
Wang Y et al (2020) Network pharmacology exploration reveals a common mechanism in the treatment of cardio-cerebrovascular disease with Salvia miltiorrhiza burge And Carthamus tinctoriusL. BMC Complement Med Ther 20:51
Liu B et al (2016) Danshen (Salvia miltiorrhiza) compounds improve the biochemical indices of the patients with oronary heart disease.Evid Based Complement Alternat Med undefined:9781715
Shi M et al (2014) Increased accumulation of the ardio-cerebrovascular disease treatment drug tanshinone in salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-D-xylulose5-phosphate reductoisomerase. Funct Integr Genomics 14:603–615
Chong CM, Su H, Lu JJ (2019) Salvia miltiorrhiza The effects of bioactive components from the rhizome of (Danshen) on the characteristics of Alzheimer's disease. Chin Med 14:19
Byun JW et al (2016) Therapeutic Effect of protocatechuic aldehyde in an in vitro model of Graves' rbitopathy. Invest Ophthalmol Vis Sci 57:4055–4062
Zhang W et al (2019) Salvianolic acid B inhibits ctivation of human primary hepatic stellate cells through ownregulation of the myocyte enhancer factor 2 signaling athway. Front Pharmacol 10:322
Liu YW, Huang YT (2014) Inhibitory effect of tanshinone IIA on rat hepatic stellate ells. PLoS ONE 9:e103229
Su CY et al (2015) Salvia miltiorrhiza: Traditional medicinal uses, chemistry, and pharmacology. Chin J Nat Med 13:163–182
Du GH, Qiu Y, Zhang JT (1995) Protective effect of salvianolic acid A on myocardial ischemia-reperfusion injury in rats. Acta Pharm Sinica 10:731–735
Du GH, Zhang JT (1995) The improving effect and mechanism of salvianolic acid A on learning and memory dysfunction induced by cerebral ischemia-reperfusion in mice. Acta Pharm Sinica 03:184–190
Liu CD et al (2021) Salvianolic acid A prevented cerebrovascular endothelial injury caused by acute ischemic stroke through inhibiting the Src signaling pathway. Acta Pharmacol Sin 42:370–381
Zhang DD et al (2018) Edaravone attenuates oxidative stress induced by chronic cerebral hypoperfusion injury: role of ERK/Nrf2/HO-1 signaling pathway. Neurol Res 40:1–10
Sarti C et al (2002) Cognitive impairment and chronic cerebral hypoperfusion: What can be learned from experimental models. J Neurol Sci 203:263–266
Lithfous S, Dufour A, Després O (2013) Spatial navigation in normal aging and the prodromal stage of Alzheimer's disease: insights from imaging and behavioral studies. Ageing Res Rev 12:201–213
Mirsaeed-Ghazi F et al (2021) Astaxanthin Decreases Spatial Memory and Glutamate Transport Impairment Induced by Fluoride. Iran J Pharm Res 20:238–254
Du J et al (2013) Mitochondrial bioenergetic deficits in the hippocampi of rats with chronic ischemia-induced vascular dementia. Neuroscience 231:345–352
Castaño Guerrero Y et al (2013) Changes in oxidative metabolism and memory and learning in an cerebral hypoperfusion model in rats. Neurología 28:1–8
Tanaka K et al (2002) Chronic Cerebral Hypoperfusion Induces Striatal Alterations Due to the Transient Increase of NO Production and the Depression of Glutathione Content. Neurochem Res 27:331–336
Sriram CS et al (2016) Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1. Physiol Behav 154:135–144
Annaházi A et al (2007) Pre-treatment and post-treatment with alpha-tocopherol attenuates hippocampal neuronal damage in experimental cerebral hypoperfusion. Eur J Pharmacol 571:120–128
Prins ND, Scheltens P (2015) White matter hyperintensities, cognitive impairment and dementia: an update. Nat Rev Neurol 11:157–165
Hung IL et al (2015) Chinese Herbal Products for Ischemic Stroke. Am J Chin Med 43:1365–1379
Schwanhausser B et al (2011) Global quantification of mammalian gene expression control. Nature 473:337–342
Kawai T et al (2001) Lipopolysaccharide Stimulates the MyD88-Independent Pathway and Results in Activation of IFN-Regulatory Factor 3 and the Expression of a Subset of Lipopolysaccharide-Inducible Genes. J Immunol 167:5887–5894
Kapinya K et al (2000) Temporary changes of the AP-1 transcription factor binding activity in the gerbil hippocampus after transient global ischemia, and ischemic tolerance induction. Brain Res 872:282–293
Li X et al (2020) Ginkgo diterpene lactones inhibit cerebral ischemia/reperfusion induced inflammatory response in astrocytes via TLR4/NF-κB pathway in rats. J Ethnopharmacol 249:112365
Yu L et al (2022) Formononetin protects against inflammation associated with cerebral ischemia-reperfusion injury in rats by targeting the JAK2/STAT3 signaling pathway. Biomed Pharmacother 149:112836

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Salvianolic acid A improves chronic cerebral ischemia through TLR4/MAPKs/AP-1 signaling pathway

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Introduction

Materials And Methods

Results

HPLC and ¹H-NMR of SAA

Effects of SAA on cognitive dysfunction and hippocampal pathological changes in rats

Changes of differential genes in rats before and after SAA treatment

Effect of SAA on expression of related proteins after chronic cerebral ischemia

Effect of SAA on expression of IL-1β, IL-12 and IL-6 in hippocampus

Discussion

Declarations

References

Status:

Version 1

Salvianolic acid A improves chronic cerebral ischemia through TLR4/MAPKs/AP-1 signaling pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

HPLC and 1H-NMR of SAA

Effects of SAA on cognitive dysfunction and hippocampal pathological changes in rats

Changes of differential genes in rats before and after SAA treatment

Effect of SAA on expression of related proteins after chronic cerebral ischemia

Effect of SAA on expression of IL-1β, IL-12 and IL-6 in hippocampus

Discussion

Declarations

References

Status:

Version 1

HPLC and ¹H-NMR of SAA