Abstract: Background and Objective: Ligusticum Chuanxiong Hort (LC) is often prescribed with borneol (BO) for a better effect in treating ischemic stroke in Asia. However, the mechanism of their combination is unclear till now. This study was aim to explore the synergy between BO and extract of LC (ELC) in protecting cortex and striatum against ischemia attack. Materials and Methods: The rats were divided into sham, model, ELC, BO and ELC+BO groups. Four-vessel occlusion surgery was employed for rat global cerebral ischemia/reperfusion (GCIR) model. After the treatment, the microcirculation, anti-oxidative ability, apoptosis index (AI), levels of apoptosis-related genes, intracellular [Ca2+] and autophagy-related proteins expressions were measured respectively. Results: In cortex, the superiority of ELC was on anti-oxidation, while that of BO was on improvement of brain microcirculation. Both of them inhibited Ca2+ overload and apoptosis in cortex via regulating p53, caspase-3, Bcl-2 and Bax. In striatum, ELC had the superiorities in regulating AI, mTOR, LC3 II/I, besides their common modulation on p53, Beclin 1 and [Ca2+]. Surprisingly, the combined group produced some new targets, including ULK1 in cortex and including CAT, GSH-Px, MDA, iNOS, NO, Bcl-2, Bax, caspase-3 and ULK1in striatum. Conclusion: The superiorities of ELC against cerebral ischemia were in inhibiting oxidation and apoptosis and promoting autophagy, while those of BO were in improving microcirculation and autophagy and inhibiting apoptosis. Their combinative therapy brought some new mechanisms in treating brain ischemia.
INTRODUCTION
Brain ischemia might arise in any cerebral region in terms of difference of pathogenesis. Ischemic stroke, as the most common stroke, is usually accompanied with damage within cortex and striatum and even accounts for up to 87% of all stroke attacks1,2. Although thrombolytic therapy is considered as a regular treatment in clinic, unfortunately, it may induce much more serious damage, such as ischemia-reperfusion injury if there is a wrong choice on therapeutic window or dose3. So, how to protect neurons in the process of treating cerebral ischemia is a critical issue in neuroscience research.
Ligusticum Chuanxiong Hort (LC) is a very famous traditional medicine in Asia for its satisfied outcome in treating cerebrovascular disease, specially ischemic stroke4-6. Our previous research suggested that tetramethylpyrazine, the most important ingredient of LC, had the bioactivities of protecting brain ischemia via anti-oxidation, inhibition of apoptosis, reduction of [Ca2+]i and promotion of angiogenesis7. However, the content of tetramethylpyrazine in LC was8 <0.1 μg g–1. Then, the human oral dose of tetramethylpyrazine is no more than 2.0 μg/person/day according to its content. Obviously, the effect of tetramethylpyrazine in LC is limited and it is possible that the brain protective effect of LC is from the other ingredients with high contents. In fact, some other constituents in LC had been identified and verified to have anti-ischemic effect separately, such as polysaccharides, ferulic acid, cnidilide and ligustilide9-13. But, the mechanism and characteristics of LC, as a whole, in treating cerebral ischemia are still unknown. Borneol (BO), known as a bicyclic monoterpene compound, is frequently prescribed for CNS disorder, including stroke, cerebral edema, dementia and cerebritis14,15. For decades of years, BO is always regarded only as a regulator of opening blood-brain barrier (BBB) in exploring the synergic mechanism between BO and the other drugs in improving brain function16. However, recent reports indicated that BO could attenuate cerebral ischemia by itself, including reducing oxidative injury, inflammation reactions, brain infarction area and apoptosis17-20. Apparently, the advantage of combinative application between LC and BO is not only in pharmacokinetics behavior, but also in pharmacodynamics synergy, which has been also confirmed by many previous studies. Huang reported that the combinative application of LC and BO exhibited a better protection against brain ischemia than their single-agent therapies21. And the mechanism of combinative advantage was concerned with reduction of neurotoxicity, enhancement of anti-oxidation and regulation of NO level22. Our previous research also found that BO increased the therapeutic effect of LC with a region-specific manner against brain ischemia and the best dose of BO was 0.08 g kg–1 for cortex and striatum23. Meanwhile, the combinative advantage between them was displayed on ameliorating the excitotoxicity of brain ischemia via reducing content of glutamate and enhancing that of γ-aminobutyric acid in brain24. Wang25 also confirmed that the microemulsion of LC and BO combination markedly reduced water content, the cerebral infarction area levels of MDA and NO in cerebral tissue and elevated the activity of SOD. In vitro study found that their commercial combination, named Suxiao Jiuxin pill, had an obvious inhibitory effect on the vasoconstriction with both endothelium-dependent and -independent manner26. Moreover, the clinic study also verified the combination of LC and BO had better therapy, including the increase of velocity and volume of blood flow in both basilar artery and vertebral artery27. Apparently, there were increasing evidences suggesting the advantage on their combinative usage. Apparently, although the synergic effective between LC and BO is clear, the potential mechanism of their co-therapy is unknown except anti-oxidative injury.
Presently, it is widely accepted that necrosis, apoptosis and autophagy are three different fates for injured neurons. Necrosis is usually accompanied with inflammation reaction and cell swelling. Apoptosis is triggered by apoptosis-related genes and usually along with pycnosis cell, deeply stained nuclei and appearance of apoptotic bodies without inflammation. Autophagy is regarded as a stress response to nutrient deprivation. This response is also a critical process to selectively remove disables cellular components, excess organelles, exogenous agents and protein aggregates with the aim of keeping homeostasis, promoting differentiation, maintaining development of cell28. Apparently, a certain degree of autophagy is in favor of the well-being of the cells29. The promotion of autophagy contributes not only to survival of neurons attacked by ischemia, but also to neurogenesis via clearance of toxic metabolites and supplement of alternative energy source30,31.
In the present study, the superiority of combining BO and extract of LC (ELC) in attenuating brain ischemia was explored. Additionally, considering that the region-specificity was another important characteristic in the combination, focused on the brain regions of cortex and striatum after global cerebral ischemia/reperfusion (GCIR) injury. The above two brain regions are usually regarded as the most vulnerable tissues to clinical ischemic stroke.
MATERIALS AND METHODS
This study was performed at the Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine from October, 2018 to August, 2019.
Both LC and BO were obtained from Nanjing Pharmaceutical Co., Ltd. (Nanjing, China). LC3 I/II, ULK1 and Beclin1 primary antibodies were from Cell Signaling Technology company (MA, USA), BNIP3 primary antibody was from Abcam (Cambridge, UK), mTOR and pAMPK primary antibodies were from Santa Cruz (CA, USA). Anti-β-actin primary antibody was from Bioworld Technology (MN, USA). Goat anti-rabbit secondary antibody was from Boster Biological Technology (Wuhan, China). The iScript cDNA Synthesis Kit was obtained from BIO-RAD Ltd., (CA, US). The SYBR Premix Ex TaqTM kit was purchased from Takara Bio Inc. (Shiga, Japan). Fluo-3/AM was from Biotium (CA, USA). TUNEL assay kit was from Roche Diagnostics Corp. (Indianapolis, IN). SOD, CAT, GSH-Px, MDA, ROS, iNOS and NO kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Trizol reagent from In vitro gen (Carlsbad, CA).
Preparation of ELC: The extraction and purification of LC were performed as previous report with minor modification32. After immersed in 1.0 L water for 30 min, 100.0 g of LC was extracted in boiling water twice and the exacted solutions were mixed together. After concentrated, it was diluted by ethanol of four times volume. The filtrate was collected after stored at 5°C overnight and the contained ethanol in filtrate was recovered by a rotary vacuum evaporator. The extract was diluted into 1.0 L by saline solution for the following experiments.
Supplementary file
HPLC analysis of ELC: The HPLC analysis was performed on 1100 series HPLC (Agilent Technology, Santa Clara, CA) equipped with a Agilent ZORBAX Eclipse XDB-C18 column (4.6 mm×250 mm id, 5 μm particle size). The mobile phase comprised 1% acetic acid in water (A) and methyl alcohol (B). The gradient flow was as follows: 0-15 min, 90-100% B. The analysis was operated at a flow rate of 1.0 mL min–1 and detected at 280 nm. The injection volume was 5 μL and the column temperature was maintained at 30°C. The representative chromatogram of ELC as shown in Fig. 1.
Animals and surgical procedure: Clean grade healthy SD rats (sex in half) were obtained from Slac Laboratory Animal Co., Ltd., (Shanghai, China), maintained in the SPF grade animal center of Nanjing University of Chinese Medicine and grouped according to Fig. 2. The light/dark cycle was 12 h and the temperature and humid were maintained at 25°C and 55% respectively. All animal procedures were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine and all operations abided by the Guide for the Care and Use of Laboratory Animals (8th edition).
| Fig. 1: | Representative chromatogram of ELC |
A: Ligustrazine, B: Senkyunolide I, C: Senkyunolide A, D: Ligustulide |
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| Fig. 2: | Flow chart of the present research |
The rat GCIR model was achieved by the surgery of four-vessel occlusion23. The rats were anesthetized by 2.5% isoflurane using a R540IP anesthesia instrument (RWD Life Science Inc., Guangdong, China) before their bilateral vertebral arteries were carefully electrocoagulated to block the blood flow. Then their bilateral common carotid arteries (CCAs) were separated. Twenty four hours later, ischemic injury of the whole brain was achieved by clipping CCAs for 60 sec with the assistance of inhalation anesthesia. And then, the clips were removed for reperfusion. The rats which lost their miosis and limb reflexes were chosen for this study. The rats in sham group were prepared by the same operation except ischemic damage. The temperatures of the rats were maintained at 37±0.5°C throughout the surgical operation.
Group intervention: As shown in Fig. 2, model rats were assigned in to 4 groups of model, ELC (1.0 g kg–1), BO (0.08 g kg–1) and ELC+BO (1.0 g kg–1 of ELC+0.08 g kg–1 of BO) by random number table method. The dose of ELC was based on its clinic prescription and that of BO was obtained according to our previous research23. The rats were orally treated according to their groups respectively once a day for a week. The sham and model control groups were administrated with physiological saline. Seven days later, the following detections were carried out at the laboratory.
Measurements of microcirculation: The procedure of measuring the microcirculation of cortex and striatum was done according to our previous report24. In this study, only cortex and striatum were chosen to detect the tissue microcirculation with the assistance of the laser Doppler probe of MP100 physiological recording system (BIOPAC Systems, Inc., CA, USA). The rat was anesthetized by 2.5% halothane during the whole procedure.
Measurements of oxidative stress: The rat was sacrificed by decapitation and its brain was taken out immediately. The cortex and striatum tissues were separated carefully. Tissue homogenates of cortex and striatum were prepared as our previous research24. The supernatant was used for the measurements of SOD, CAT, GSH-Px, MDA, ROS, iNOS and NO concentrations by the assay kits according to their protocols provided by their manufacturers.
Apoptosis index (AI) measurement: TUNEL examination was used to evaluate apoptotic degree of ischemic neurons as our previous research7. Rat was deeply anesthetized by 2.5% halothane and then perfused with NS and 4% paraform aldehyde successively. After cortex and striatum were separated from the brain, they were fixed with paraformaldehyde. The brain tissue was placed in paraffin and cut into sections (5 μm) after dehydration. The operation of TUNEL staining was performed with the instruction of TUNEL assay kit (Roche Diagnostics Corp., Indianapolis, IN). The tissues were observed under a microscope. The cells stained with brown were positive neurons of apoptosis. AI was the ratio of positive neurons to all neurons from three random visual fields and used to evaluate the degree of apoptosis.
Measurement of apoptosis-related genes: The levels of apoptosis-related genes were measured as our previous research7. After the separation of cortex and striatum from the whole brain, Trizol reagent was employed to extract the total RNA. After quantitative determination of total RNA, the RNA was reverse-transcribed via iScript cDNA Synthesis Kit. The SYBR Premix Ex TaqTMkit was used to conduct real-time PCR and 2-ΔΔCt, as the comparative threshold, was used to assay the contents of apoptosis related genes. The primer sequences of p53, caspase-3, Bcl-2, Bax and GAPDH and conditions of amplification were designed as our previous report7.
Expressions of autophagy-related proteins: The expressions of autophagy-related proteins were measured as our previous research7. After protein quantification, the denatured protein was diluted with loading buffer, separated via SDS-PAGE and transferred to a PVDF membrane. After non-specific binding sites were blocked, the membrane was incubated in the following primary antibodies at 4°C overnight: pAMPK, mTOR, ULK1, LC3 I/II, Beclin1 and BNIP3. Anti-β-actin was used for normalization. After washed by TBST for three times, the membrane was incubated in secondary antibody and washed again. The blots were captured by ImageQuant LAS4000 mini (GE Healthcare, PA, USA).
Measurement of neuronal [Ca2+]i: The [Ca2+]i was measured as our previous research7. After the cortex and striatum were separated from the whole brain in the ice-cold DMEM solution filled with 95% O2 and 5% CO2, tissue slices of 300 μm thickness were prepared using a tissue microtome. The slice was laid on a cover glass and incubated in DMEM solution filled with O2 for 0.5 h and Fluo-3/AM was added with the final concentration of 5.0 μM. After washed with D-Hanks, the slice was ready to be used for fluorescence detection under a laser scanning confocal microscope (Leica TCS-SP5, Solms, Germany) with 488 nm of excitation wavelength. The emission wavelength was within 505-530 nm. [Ca2+]i was denoted by the fluorescence intensity.
Statistical analysis: GraphPad Prism 5.0 and SPSS 13.0 were used for drawing charts and data analysis respectively. The data was expressed as mean±SD. One-way analysis of variance and Tukey’s multiple comparison were employed for significance analysis. P with the value <0.05 was regarded as significantly difference.
RESULTS
Microcirculation measurement: Compared to sham group, GCIR injury markedly reduced microcirculations in both cortex and striatum which confirmed the severe damage by ischemia. Administration of ELC or BO increased the microcirculation in cortex. However, neither of them exhibited improvement in striatum.
| Fig. 3(a-b): | Synergic effect of ELC and BO on microcirculations in (a) Cortex and (b) Striatum of GCIR rats |
Mean±SD, n = 6, BPU: Blood perfusion units, oral dosages of ELC and BO were 1.0 and 0.08 g kg–1 respectively, ##p<0.01 vs. sham group, *p<0.05, **p<0.01 vs. model group, &p<0.05 vs. ELC+BO group |
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Interestingly, the combination of ELC and BO not only produced significant enhancement on striatum microcirculation, but also showed a better improvement than ELC monotherapy in cortex. The results, as shown in Fig. 3, indicated their synergic effect on microcirculations in the two brain regions.
Antioxidant activity: The results in cortex were shown in Fig. 4. Compared to sham group, GCIR injury induced the formation of oxidative stress reaction, including the decreases of SOD, CAT, GSH-Px and the increases of MDA, ROS, iNOS and NO in cortex. ELC exhibited excellent anti-oxidative bioactivities on all of above indices, while BO did not show any marked improvement except iNOS. Moreover, their combination showed more powerful modulation on GSH-Px than BO group.
Figure 5 illustrated the results in striatum. Compared to the sham group, GCIR injury induced the same changes as in cortex. However, in this area, both ELC and BO did not show any improvement against oxidative injury compared to model group.
| Fig. 4(a-g): | Synergic effect of ELC and BO on anti-oxidation in cortex of GCIR rats, (a) SOD, (b) CAT, (c) GSH-Px, (d) MDA, (e) ROS, (f) iNOS and (g) NO |
Mean±SD, n = 10, ##p<0.01 vs. sham group, *p<0.05, **p<0.01 vs. model group, &p<0.05 vs. ELC+BO group |
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| Fig. 5(a-g): | Synergic effect of ELC and BO on anti-oxidation in striatum of GCIR rats, (a) SOD, (b) CAT, (c) GSH-Px, (d) MDA, (e) ROS, (f) iNOS and (g) NO |
Mean±SD, n = 10, #p<0.05, ##p<0.01 compared to sham group, *p<0.05 compared to model group |
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| Fig. 6(a-c): | Synergic effect of ELC and BO on AI in cortex and striatum of GCIR rats, (a) TUNEL staining images of each group (×200). The cells stained with brown were positive neurons of apoptosis, (b) AI column charts of each group in cortex, n = 8, (c) AI column charts of each group in striatum |
Oral dosages of ELC and BO were 1.0 and 0.08 g kg–1, respectively, n = 8, ##p<0.01 vs. sham group, **p<0.01 vs. model group, &p<0.05, &&p<0.01 vs. ELC+BO group |
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But their synergic treatment induced the enhancement of CAT and GSH-Px and the reduction of MDA, iNOS and NO.
AI measurement: As shown in Fig. 6, the neurons stained with dark brown were regarded as apoptotic positive cells. Compared to sham group, GCIR injury induced the formation of large scale of apoptosis and increased AI in cortex and striatum areas. Administration of ELC markedly reduced AI in both of the two areas, while BO inhibited the formation of apoptosis only in cortex. Additionally, their combination not only showed better improvements than BO group in cortex, but also exhibited better therapy than both monotherapies of ELC and BO in striatum.
Apoptosis-related genes levels: Compared to the sham group, GCIR injury induced the formation of apoptosis, along with reducing Bcl-2 and enhancing Bax, p53 and caspase-3. Interestingly, the improvement from ELC and BO exhibited a region-specific manner. In cortex, the administrations of ELC, BO and their combination had the ability to increase Bcl-2 and decrease Bax, p53 and caspase-3. The results indicated that both of the drugs had the bioactivity of inhibiting apoptosis and protecting neurons. While, in striatum, both ELC and BO only decreased p53 level. Compared to the monotherapy of BO, their combination displayed more powerful modulation on p53, Bcl-2 and Bax genes. Moreover, their combination brought some new targets on anti-apoptosis in striatum, including the regulations on Bcl-2, Bax and caspase-3, which displayed their synergic effect more clearly. The results were shown in Fig. 7.
Autophagy-related proteins levels: Compared to the sham group, the model group exhibited the formation of autophagy via changing expressions of autophagy-related proteins, including increase of Beclin1 in cortex and decreases of mTOR in both of the two brain regions.
| Fig. 7(a-b): | Synergic effect of ELC and BO on expressions of Bcl-2, Bax, p53 and caspase-3 genes in (a) Cortex and (b) Striatum of GCIR rats |
Mean±SD, n = 5, #p<0.05, ##p<0.01 vs. sham group, *p<0.05, **p<0.01 vs. model group, &p<0.05, &&p<0.01 vs. ELC+BO group |
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Interestingly, ELC and BO had the ability to strengthen autophagy with different cmechanisms and even the same drug had different targets in different brain regions with a region-specific manner.
In cortex, ELC enhanced the expressions of pAMPK, Beclin1 and LC3 II/I and reduced that of mTOR. While BO increased the levels of Beclin1 and LC3 II/I. The results indicated that both ELC and BO further improved protective autophagy. Their combination had better improvement and even produced a new target of ULK1. Compared to ELC group, the combination further increased the expressions of pAMPK, ULK1 and LC3 II/I. In comparison to BO group, the combination further decreased the expression of mTOR, elevated Beclin1 and pAMPK. The results were shown in Fig. 8.
In striatum, ELC had the ability to increase Beclin1 and LC3 II/I. Similarly, BO enhanced the expression of Beclin1. In addition, their combination modulated two new targets, up-regulation of ULK1 and down-regulation of mTOR. Apparently, their synergic effect on autophagy in striatum was shown clearly. The results were displayed in Fig. 9.
Neuronal [Ca2+]i: Compared to the sham group, the neuronal [Ca2+]i in the model group increased sharply, which indicated that GCIR injury induced the formation of Ca2+ overload. Both ELC and BO had the ability of reducing neuronal [Ca2+]i within cortex and striatum regions. Moreover, their combination showed better modulation than their monotherapies in both of the two brain areas. The result was shown in Fig. 10.
DISCUSSION
Ischemic stroke, as an important part making up cerebrovascular disease which is the second cause of death with the mortality rate of 10.6%, is a severe neurological disorder attracting neuroscientist’s attention worldwide33. It is well known that the ischemia cascade is initiated in the hypo-perfusion region of the brain and further widely activated in reperfusion phase of the above region34,35. In this study, the phase of hypo-perfusion occurred during the period of simultaneously b locking the blood flow of bilateral vertebral arteries and CCAs. And the reperfusion phase beginning with loosing the clips around CCAs. Oxidative stress is regarded as fundamental mechanism of ischemia/reperfusin injury. Reactive oxygen species (ROS) predominantly contributes to the early stage of GCIR injury. ROS, produced in electron transmitter chain, should be eliminated by SOD, CAT and GSH-Px to avoid damage of biomembrane by peroxide reactive36. The present study indicated that ELC or BO increased the microcirculation in cortex and their combination not only enhanced the microcirculation in striatum, but also made a further improvement in cortex. Moreover, in cortex, ELC exhibited ideal anti-oxidative effects, including the increases of SOD, CAT, GSH-Px and the decreases of MDA, ROS, iNOS and NO. In striatum, their combination increased GSH-Px and CAT and reduced MDA, iNOS and NO, although their monotherapy showed no improvement on oxidative stress in this area.
Increasing evidences confirm a mutual promotion between ROS and Ca2+ signal pathway. Dysfunction in either of the pathways might affect the other one and produce potential abnormal conditions which might induce various other disorders37. Commonly, the formations of ROS and Ca2+ were involved in cellular respiratory chain in mitochondrion and their excess productions always induce serious cytotoxicity. The activation of mitochondrial permeability transition pore is regarded as one of the important regulators to trigger the release of ROS and change mitochondrial redox38,39. Furthermore, Ca2+, known as a second messenger, also participates in cellular cascade reactions which might decide the survival or death of a cell. However, excess concentration of intracellular Ca2+, known as Ca2+ overload, may produce a series of reverse action. When Ca2+ overload arises, both endoplasmic reticulum and mitochondria are important sources of this ion40. Additionally, ischemia injury promotes the influx of Ca2+ into neuronal cells not only by the accumulation of ROS, but also by excitotoxic mechanism41. Xing42 reported that decreasing [Ca2+]i helped to ameliorate endoplasmic reticulum stress and protected cells against hypoxia-reoxygenation injury. Previous report indicated that various cytokines had the ability to activate iNOS, including exotoxins and TNF-α and the formation of NO from iNOS was related to apoptosis injury43. In the present study, both ELC and BO had the ability of reducing [Ca2+]i in cortex and striatum regions. Moreover, their combination showed a better improvement than their monotherapies in both of the two brain areas. Similarly, there was clear region-specificity in modulating iNOS and NO. In cortex, ELC decreased both iNOS and NO. While, in striatum, only their combination had the ability to reduce iNOS and NO instead of their monotherapy.
It has been verified that the Bcl-2 family play a key role in regulating cascade reactions of cellular apoptosis and the ratio of Bax and Bcl-2 is commonly regarded as a key factor to decide the appearance of apoptosis44. Moreover, the cascade reaction of caspase family, activated by influx of Ca2+, is another critical factor inducing neuronal apoptosis.
| Fig. 8(a-b): | (a-b) Synergic effect of ELC and BO in regulating autophagy-related proteins levels in cortex of GCIR rats |
Means±SD, n = 5, ##p<0.01 vs. sham group, *p<0.05, **p<0.01 vs. model group, &p<0.05 vs. ELC+BO group |
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| Fig. 9(a-b): | (a-b) Synergic effect of ELC and BO in regulating autophagy-related proteins levels in striatum of GCIR rats |
Mean±SD, n = 5, #p<0.05 vs. sham group, *p<0.05 vs. model group, &p<0.05 vs. ELC+BO group |
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| Fig. 10(a-d): | Synergic effect of ELC and BO in regulating neuronal [Ca2+]i in cortex and striatum of GCIR rats, (a, b) Laser confocal microscope in cortex and striatum regions and (c, d): Column charts of the results from cortex and striatum, respectively |
Means±SD, n = 8, 1-5 represented the groups of sham, model, ELC, BO and ELC+BO respectively, RFU: Relative fluorescence unit, level of fluo 3/AM reflected the intracellular concentration of Ca2+, ##p<0.01 vs. sham group, *p<0.05, **p<0.01 vs. model group, &p<0.05, &&p<0.01 vs. ELC+BO group |
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The activation of caspase family, particularly caspase-3, is supposed to be the strongest criteria of the formation of apoptosis because caspases reaction is not found in necrotic deatharea45,46. Because of the evidence, caspase-3 is usually considered to be closely related to apoptosis and even applied as a biomarker of this programmed cell death47. p53 is mainly expressed in the mitochondria and regarded to contribute to cell apoptosis48. In this study, a clear region-specific manner was also found on apoptosis modulation. ELC reduced apoptosis in both of the two areas, while BO only inhibited it in cortex. In a further mechanism research, both of the drugs inhibited apoptosis via down-regulating caspase-3, Bax and p53 and up-regulating Bcl-2 within cortex. But they only decreased p53 in striatum. However, their combination indicated some new targets on anti-apoptosis in striatum, including Bcl-2, Bax and caspase-3.
Autophagy, as a way of cellular renewal, is different from apoptosis and necrosis. Usually, it is activated by hunger, protein aggregation, apoptosis, hypoxia and pathogenic infection and help to promote the process of cellular reconstruction, regeneration and repair via regulating related pathways, such as pAMPK-mTOR-ULK1 and BNIP3-Beclin149-52. Oxidative injury is considered to be a trigger of autophagy. It induces phosphorylation of AMPK and then reduces the level of mTOR53. Then deficiency of mTOR further modulates the expressions of its downstream proteins, such as ULK1, which contributes the formation of autophagosome by transforming LC3 I to LC3 II54. Hypoxia is believed to be another trigger of autophagy by mediating the BNIP3-Beclin1 signaling pathway, which, similar to above pAMPK-mTOR-ULK1, finally produces the autophagosome after the transformation between LC3I and LC3 II55. The present study confirmed that ischemia might trigger protective autophagy, which was similar to previous reports56,57. Both ELC and BO further enhanced autophagic degree on the basis of ischemic injury in the two brain areas. ELC increased the expressions of Beclin1, LC3 II/I in both of the areas. Furthermore, the level of pAMPK was enhanced with the treatment of ELC in cortex. The targets of BO in regulating autophagy were Beclin1, mTOR and LC3 II/I in cortex and only Beclin1 in striatum. Similarly, their combination still exhibited a better modulation on autophagy with the new targets of ULK1 in cortex and striatum and mTOR in striatum.
Interestingly, there may be interplay between autophagy and apoptosis. It was found that ischemic injury could be protected by the activation of autophagy via modulating pAMPK-mTOR pathway and, reversely, the inhibition of the autophagy pathway aggravated apoptotic damage58. Since it is totally different cellular fate between harmful apoptosis and protective autophagy, which will make the choice? A recent research indicated that the concentration of intracellular Ca2+ might act as a switch between apoptosis andautophagy59. In the present study, the discovery, that the combination treatment transformed apoptosis to autophagy accompanying with the decrease of [Ca2+]i, confirmed the switch role of Ca2+ once again. On account of the high homology in gene makeup between rat and human, the findings in this study discovered the mechanism of combinative prescription of LC and BO for attenuating ischemic stroke.
CONCLUSION
There were synergic effects between ELC and BO in protecting cortex and striatum followed GCIR injury. The superiorities of ELC were in inhibiting oxidation and apoptosis and promoting autophagy, while those of BO were in improving microcirculation and autophagy and inhibiting apoptosis. Their combinative therapy showed some new mechanisms in treating brain ischemia.
SIGNIFICANCE STATEMENT
The study explored the synergic mechanism between ELC and BO against ischemic injury in cortex and striatum regions. ELC and BO had their respectively superiorities in the treatment. The study provides future researchers with a new perspective to explore the synergic effect between the treatments for cerebral ischemia.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (81973726.81573713), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Qinglan Project of Jiangsu Province (2017), Jiangsu Provincial Key Construction Laboratory (No. SuJiaoKe [2016]8), the Jiangsu Overseas Visiting Scholar Program for University Prominent Young and Middle-aged Teachers and Presidents (2018).