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Batatasin-III Regulates the NO Production and Mitochondrial Membrane Potential in Cerebral Vascular Endothelial Injury: in-vivo and in-vitro Study

Yanzhu Huang, Ling Li , Shanshan Huang , Yafeng Zhang, Qing Zhang, Zhimei Li, Yue Cao, Wenjuan Yu, Shuhua Tong, Qiang Zhang
Journal of Food and Nutrition Research. 2021, 9(10), 499-507. DOI: 10.12691/jfnr-9-10-1
Received August 28, 2021; Revised October 02, 2021; Accepted October 11, 2021

Abstract

Microvascular endothelial cell injury causes cerebral injury. Present study evaluates the protective effect of batatasin-III against cerebral microvascular endothelial cell (EC) injury by in-vivo and in-vitro study. Endothelial cells were isolated from rats and exposed to oxygenglucose deprivation/reperfusion (OGD/R) condition to induced endothelial injury cell. These cells were treated with batatasin-III (5, 10 and 20 μM) during the 4h period of OGD insult and cells were further incubated for 24 h under the normal condition. Cell viability was estimated by MTT assay and LDH release, mitochondrial membrane potential and level of NO was estimated in (OGD/R) exposed ECs. Apoptosis of ECs was estimated by Hoechst 33258 and expression of apoptotic protein by western blot assay in ECs. Moreover in vivo study was performed to determine the effect of batatasin-III on cerebral ischemia induced brain injured rats. Result of investigation reveals that treatment with batatasin-III ameliorates the cell viability and percentage of LDH release in the OGD/R induced EC injury. Expression of apoptotic protein and integrity of mitochondrial membrane was alleviated in batatasin-III treated (OGD/R) induced EC injury. Level of NO and apoptosis of ECs was reduced in batatasin-III treated group than OGD/R group of ECs. Moreover, infract size, apoptosis of neuronal cells and pathological changes were ameliorated in batatasin-III treated group than MCAO group of rats. In conclusion, data reveals that batatasin-III treatment reduces the cellular apoptosis by reduces the oxidative stress and improving mitochondrial membrane potential in microvascular endothelial cell injury and improved the cerebral injury.

1. Introduction

Vascular endothelial cells (VEC) which contribute to the maintenance of central nervous system function 1. VEC present in the microvessels of brain releases several neurochemicals such as endothelial-derived contracting factors, hyperpolarization factor, prostacyclin and nitric oxide, which regulates the cerebral blood flow 2. Blood brain barrier structure is formed due to formation of tight junction between endothelial cells. It limits transfer unwanted or toxic material to reach to the brain. Moreover, modulation of NO release and platelet adhesion regulates the cerebral EC maintained anti-inflammatory and antithrombotic active surface 3. Cerebral ischemia or other toxic stimuli enhances the neuronal injury by disturbing the integrity of blood brain barrier and neuronal inflammation due to EC injury 4.

In neurodegenerative disorders, evidence suggests that loss of cortical function occurs due to necrosis or apoptosis in the ECs leads to vascular degeneration 5. Integrity of membrane lost as necrosis or apoptosis of ECs which causes cellular organelle damage. Literature reveals that apoptosis of EC occurs due to reduction in the generation NO, which is generated from endothelial nitric oxide synthetase 6. This oxidative stress production alters the mitochondrial membrane potential which activates the caspase 2 and 6 enzymes 7. Caspase cascade activation stimulates the apoptosis of ECS.

Dihydrostilbenes are the polyphenols known for its antioxidant, anti-inflammatory and GABA aminergic activities 8. Batatasin-III is chemically a dihydrostilbene isolated from Dendrobium draconis Rchb.f 9. Batatasin-III known for its potential antioxidant, anticancer and anti-diabetic activity 10, 11, 12. Derivatives of batatasin shows anti-inflammatory property by inhibiting the COX2 enzyme and thereby reducing cytokine and prostaglandin production 13. Thus, presented report evaluates the protective effect of Batatasin-III against cerebral vascular endothelial injury.

2. Material and Methods

2.1. Animals

Sprague Dawley rats were kept under a standard protocol as per the guideline. All the protocol was approved by institutional animal ethical committee of Tongji Hospital of Huazhong University of science and technology, Wuhan, China (IAEC/TH-HUST/2019/08).

2.2. Chemicals

Batatasin-III was generously supplied from Chinese Academy of Sciences, Beijing, China. Culture media and JC-1 assay kit was purchased from Thermo Fisher Scientific Inc., USA. Lactate Dehydrogenase (LDH) Assay Kit was procured from abcam LTD, USA. Primary and secondary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

2.3. Preparation of Primary Microvascular EC

Collagenase/dispase based digestion protocol was used to isolate the microvascular EC from adult rats 14. Cerebrum was isolated from each animal and suspend the same into antibiotic-antimycotic solution (5%) containing collagenase/dispase (1 mg/ml) solution in M199E for the dissociation at 37°C for 2 h. Tissue was homogenized using tissue homogenizer. Pellet was suspended in HEPES buffer and further centrifuge at 4°C for 20 min at 4,000 g. Dulbecco’s phosphate saline containing percoll (45%) of colloidal silica gradient solution was used to resuspend the vascular pellet and centrifuge it at 10°C for 20 min at 20,000 g. Homogenate was centrifuge three times and thereafter upper band which consists of single cells were removed from it. Culture media containing M199E with supplementation of EC growth supplement (20 Ìg/ml), heparin (90 Ìg/ml), L-glutamine (2 mM) and 20% of fetal bovine serum was used to resuspend the pellet. Cells were cultured for 1 h at 37°C and under the microscope endothelial cells were identified for the further study.

2.4. In-vitro Oxygen-glucose Deprivation/Reperfusion Induced Endothelial Injury Cell (OGD/R) Model and Batatasin-III Treatment

Cells were cultured as per the earlier mention culture procedure and later Earle’s balanced salt solution was added in the cell culture in replacement of medium 15. OGD insult was initiated by placing the cells with Anaero Pack in Anaero container for 4h. Further cells were cultured for 24 h in the fresh medium at 37°C to terminate the OGD. Cells were treated with Batatasin-III (5, 10 and 20 μM) during the 4h period of OGD insult and cells were further incubated for 24 h under the normal condition.

2.5. Determination of cell Viability

Cell viability was determined by MTT assay as per previously reported study 16. Cells were treated with Batatasin-III during the period of induction OG insult as per earlier description and cells were incubated at 37°C with MTT solution for 4 h after the culture the cells under normal condition for 24h. Optical density was determined after replacement of supernatant solution with DMSO (100 µl) at 570 nm.

2.6. Determination of LDH Release

Cells subjected to ODG insult treated with Batatasin-III as per earlier description in the study. LDH kit was used to estimate the release of LDH from cells as per the direction of manufacturer of kit. The absorbance of reaction mixture was estimated at a wavelength of 450 nm.

2.7. Determination of Mitochondrial Membrane Potential

Cells subjected to ODG insult treated with Batatasin-III as per earlier description in the study. JC-1 assay kit was used to estimate the mitochondrial depolarisation. Cells were treated with 10 µg/ml JC-1 solution and incubate it in the dark for 20 min at 37°C. Later cells were washed with PBS and fluorescence microscope was used to determine the absorbance of JC-1 monomer at 460 and 530 nm wavelength and JC-1 polymer at 520 and 590 nm wavelength. Normal mitochondria was consider in the cells showing red polymeric fluorescence.

2.8. Determination NO Level

Cells subjected to ODG insult treated with Batatasin-III as per earlier description in the study. Cells were treated with trypsin (0.125%) for digestion after removing the culture. Further cells were crushed using ultrasonication and centrifuge the same at 1600 g at 4°C. Precooled microcentrifuge tube was used to collect the supernatant. Level of NO, MDA and SOD was determined as per the direction of manufacturer of kits.

2.9. Hoechst 33258 Staining

Cells subjected to ODG insult treated with Batatasin-III as per earlier description in the study. Paraformaldehyde (4%) was used to fix the cells and cells were treated with Hoechst 33258 working solution (1 μg/ml) and incubate it at room temperature for 30 min. fluorescence microscope was used to capture the image of cells at 352 and 461 excitation and emission wavelength.

2.10. Western Blot Assay

Extraction of total protein from cells was performed after treatment with protein lysis buffer. Total protein concentration was estimated using a DC Protein Assay. Isolated protein was separated using a sodium dodecyl sulphatepolyacrylamide gel (10%) and filtered with a polyvinylidene difluoride membrane. The membrane was then treated with 5% fresh nonfat dry milk to block the reaction, incubated at 4°C overnight with primary antibodies, such as Bcl-2 (dilution 1:500), caspase-3 (dilution 1:500), cleaved caspase-3 (dilution 1:2000), PARP (dilution 1:2000), cleaved PARP (dilution 1:500) and GAPDH (dilution 1:2000), and thereafter incubated with secondary antibodies. densitometric analysis of the blots was performed using Image Lab software.

2.11. In-Vivo Induction of Cerebral Ischemia

Cerebral ischemia was induced in the animal by performing MCAO using intraluminal filament model. Ischemia was induced for the period of 60 min and thereafter removal of suture was done to achieve reperfusion.

2.12. Determination Infract

Brain was isolated from each animal after sacrificing them. Brain tissue was sectioned into 3 mm section and tissue section was stained with TTC solution (0.1%). Viable region of the brain shows red formazan and infracted region was found to be remain unstained.

2.13. TUNEL Assay

Apoptosis of vascular cells was determined by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay as per reported method 17. Apoptotic cells were indicated by brown stained dots, number of apoptotic cells were observed and counted.

2.14. Estimation of Changes in the Histopathology

Isolated brain was fixed in formalin solution and further brain tissue was dehydrated by using ethanol. Tissue was seeded into paraffin and section of 5 µM thickness was sectioned from the brain tissue using microtome. H&E stain was used to stain the brain tissue and trinocular microscope was used to estimate the histopathological changes in the brain tissue.

2.15. Statistical Analysis

All data are expressed as means ± standard error of the mean (SEM; n = 6), and the statistical analysis consisted of a one-way analysis of variance (ANOVA). Post-hoc comparisons of means were carried out with Dunnett’s post-hoc test using GraphPad Prism software (ver. 6.1; San Diego, CA, USA). P values < 0.05 were considered to indicate statistical significance.

3. Results

3.1. Effect of Batatasin-III on the Viability of EC

Effect of batatasin-III was determined on cell viability and LDH release in OGD/R induced endothelial cell injury as shown in Figure 1. There was reduction in the percentage of cell viability (45%) and increase in the percentage of LDH release (52%) in the OGD/R group than control group. Percentage of LDH release and cell viability was reversed in batatasin-III treated group upto 99% and 14% respectively than OGD/R group of ECs.

3.2. Effect of Batatasin-III on the Depolarization of Membrane Potential

Mitochondrial membrane potential was assessed by determining the percentage of cells with depolarized mitochondria in OGD/R induced endothelial cell injury. There was significant (p<0.01) increase in the percentage of depolarized mitochondria in OGD/R group than control group of ECs. Batatasin-III treatment significantly (p<0.01) reduces the percentage of depolarized mitochondria in OGD/R induced endothelial cell injury (Figure 2).

3.3. Effect of Batatasin-III on the Level of NO in the ECs

Parameters of oxidative stress was assessed in batatasin-III treated OGD/R induced ECs. Level of NO (17.2 µmol/mg) and MDA (14.2 µmol/mg) was enhanced and reduces the activity of SOD upto 6.4 U/mg in ECs of OGD/R group than control group. Treatment with batatasin-III reverse the altered level of MDA and NO and activity of SOD in the OGD/R induced ECs injury.

3.4. Effect of Batatasin-III on the Apoptosis of ECs

Apoptosis of ECs was estimated by determining the percentage of apoptotic cells (Hoechst 33258 staining) and expression of caspase-3, cleaved caspase-3, PARP, cleaved PARP and Bcl-2 proteins as shown in Figure 4 A&B. Data of Hoechst 33258 staining of ECs shows increase in the percentage of apoptosis in OGD/R group than control group, treatment with batatasin-III was reduced in percentage of apoptotic cells in OGD/R induced endothelial injured cell (Figure 4A). Expression of Bcl-2, caspase-3 and PARP was reduced and increase in the expression of cleaved caspase-3 and cleaved PARP in OGD/R group than control group of ECs. There was reduction in the oxidative stress parameters in batatasin-III treated group than OGD/R group (Figure 4B).

3.5. Effect of Batatasin-III on the Infract Size and Apoptosis Ratio

Endothelial cell injury was assessed by determining the percentage of infract size and apoptosis ratio of vascular cells in the brain of batatasin-III treated cerebral ischemia rats. Percentage of infract size and apoptosis ratio of vascular cells was enhanced significantly (p<0.01) in the brain tissue of MCAO group than control group of mice. Batatasin-III treated group shows reduction in the apoptosis ratio and percentage of infract size in the brain tissue of cerebral ischemia rats (Figure 5).

3.6. Effect of Batatasin-III on the Histopathology of Brain Tissue

Histopathological changes were assessed by observing the changes in the brain tissue of batatasin-III treated MCAO induced cerebral ischemia model using H&E staining. Histopathological changes in the brain tissue of MCAO group shows karyopyknosis and vacuolation like changes in the cerebral neurons and these changes were reversed in the batatasin-III treated MCAO induced cerebral ischemia model (Figure 6).

4. Discussion

Blood brain barrier permeability is altered and causes break down of it due to in the ischemic condition to the brain tissue 18. Moreover, in cerebral ischemia main pathological feature is damage of BBB and maintenance of BBB is the major target for the treatment of cerebral ischemia. Literature suggests that injury in the microvascular endothelial cell contributes it the development of ischemic condition to the cerebral tissues, as these cells regulates the several physiological function like fibrinolysis, antithrombosis and anticoagulant activity 19. Moreover, microvascular endothelial cells are more sensitive in concern with the hypoxia or ischemia like condition 20. Thus, present report focused its study on OGD/R induced EC injury to understand the management of ischemia induced cerebral injury.

Production of free radicals and its chain reaction plays an important role in the brain damage caused by ischemic injury 21. Lipid enriched brain tissue produces lipid peroxides due to free radicals. Structure and function of cells altered significantly by the production of MDA, which is a major product of lipid peroxidation and assessment of extent of cell damage represented by the amount of MDA 22. Moreover, SOD is another parameter of assessment of oxidative stress, as it removes the free radicals. Calcium accumulation in the cell enhances the generation of NO by activating the nitric oxide synthetase enzyme. Literature suggests that peroxynitrite production enhances due to combination of NO and peroxides 23. Nitric oxide reported to enhance the glutamate release in the brain tissue which causes damage in the brain tissue 24. Data of the reported study suggest that parameters of oxidative stress enhance in the ODG/R exposed ECs and treatment with batatasin-III ameliorates the altered level of these parameters in ODG/r exposed ECs.

Mitochondria function alters in in cells exposed to ischemic condition including ECs. Bcl-2 protein involved in regulation of apoptosis pathway of mitochondria and also maintains the potential of mitochondrial membrane 25. Literature suggests that ischemic condition and decreased energy production reduces the expression of Bcl-2 protein which dysregulates the mitochondrial membrane integrity 26. These events in the EC activates the caspase pathway leads to apoptosis due to mitochondrial release of cytochrome C. Data of given report also reveals that ECs exposed to ischemic condition causes alteration in the expression of Bcl-2 protein and activates the caspase pathway due to loss of integrity of mitochondrial membrane. Treatment with batatasin-III reduces the apoptosis of ECs which is induced by OGD/R insult. Moreover result of in vivo study also supports that batatasin-III reduces the apoptosis in the neuronal cells and histopathological alteration in the brain tissue in cerebral ischemia induced neuronal injured animals.

5. Conclusion

In conclusion, result represented reveals that batatasin-III treatment reduces the cellular apoptosis by reduces the oxidative stress and improving mitochondrial membrane potential in OGD/R induced microvascular endothelial cell injury. There was reduction in the pathological changes and neuronal apoptosis in batatasin-III treated cerebral ischemia induced brain injured animals.

Acknowledgements

All the author of this manuscript are thankful to The Second Hospital of Shandong University, China for providing necessary facility to conduct the presented work.

Author’s Contribution

LL and YZ designed, performed and supervised the study and drafted the original manuscript; ZH performed the study and roughly drafted manuscript; SH, QZ and ZL contributes in statical analysis of data; YC, WY, ST and QZ contribute for experimental study and also revised the drafter manuscript.

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Published with license by Science and Education Publishing, Copyright © 2021 Yanzhu Huang, Ling Li, Shanshan Huang, Yafeng Zhang, Qing Zhang, Zhimei Li, Yue Cao, Wenjuan Yu, Shuhua Tong and Qiang Zhang

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Cite this article:

Normal Style
Yanzhu Huang, Ling Li, Shanshan Huang, Yafeng Zhang, Qing Zhang, Zhimei Li, Yue Cao, Wenjuan Yu, Shuhua Tong, Qiang Zhang. Batatasin-III Regulates the NO Production and Mitochondrial Membrane Potential in Cerebral Vascular Endothelial Injury: in-vivo and in-vitro Study. Journal of Food and Nutrition Research. Vol. 9, No. 10, 2021, pp 499-507. http://pubs.sciepub.com/jfnr/9/10/1
MLA Style
Huang, Yanzhu, et al. "Batatasin-III Regulates the NO Production and Mitochondrial Membrane Potential in Cerebral Vascular Endothelial Injury: in-vivo and in-vitro Study." Journal of Food and Nutrition Research 9.10 (2021): 499-507.
APA Style
Huang, Y. , Li, L. , Huang, S. , Zhang, Y. , Zhang, Q. , Li, Z. , Cao, Y. , Yu, W. , Tong, S. , & Zhang, Q. (2021). Batatasin-III Regulates the NO Production and Mitochondrial Membrane Potential in Cerebral Vascular Endothelial Injury: in-vivo and in-vitro Study. Journal of Food and Nutrition Research, 9(10), 499-507.
Chicago Style
Huang, Yanzhu, Ling Li, Shanshan Huang, Yafeng Zhang, Qing Zhang, Zhimei Li, Yue Cao, Wenjuan Yu, Shuhua Tong, and Qiang Zhang. "Batatasin-III Regulates the NO Production and Mitochondrial Membrane Potential in Cerebral Vascular Endothelial Injury: in-vivo and in-vitro Study." Journal of Food and Nutrition Research 9, no. 10 (2021): 499-507.
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  • Figure 1. Effect of batatasin-III on the cell viability and LDH release in OGD/R induced endothelial cell injury (Mean±SEM (n=6); ##p<0.01 than control group; **p<0.01 than OGD/R treated group)
  • Figure 2. Batatasin-III reduces the percentage of depolarized mitochondria in OGD/R induced endothelial cell injury (Mean±SEM (n=6); ##p<0.01 than control group; **p<0.01 than OGD/R treated group)
  • Figure 3. Batatasin-III ameliorates the level of NO and MDA and activity of SOD in OGD/R induced endothelial cell injury (Mean±SEM (n=6); ##p<0.01 than control group; **p<0.01 than OGD/R treated group)
  • Figure 4. Batatasin-III protects the apoptosis of ECs in OGD/R induced endothelial cell injury. A: Percentage of apoptosis of ECs by Hoechst 33258 staining; B: Relative expression of proteins such as caspase-3, cleaved caspase-3, PARP, cleaved PARP and Bcl-2 by western blot assay (Mean±SEM (n=6); ##p<0.01 than control group; **p<0.01 than OGD/R treated group)
  • Figure 5. Batatasin-III reduces the endothelial cell injury in cerebral ischemia rat model. A: Assessment of percentage of infract size; B: Assessment of apoptosis ratio by TUNEL assay (Mean±SEM (n=6); ##p<0.01 than control group; **p<0.01 than OGD/R treated group)
[1]  Beazley-Long N, Durrant AM, Swift MN, Donaldson LF. The physiological functions of central nervous system pericytes and a potential role in pain. F1000Res. 2018 Mar 20; 7: 341.
In article      View Article  PubMed
 
[2]  Sandoo A, van Zanten JJ, Metsios GS, Carroll D, Kitas GD. The endothelium and its role in regulating vascular tone. Open Cardiovasc Med J. 2010 Dec 23; 4: 302-12.
In article      View Article  PubMed
 
[3]  van Hinsbergh VW. Endothelium--role in regulation of coagulation and inflammation. Semin Immunopathol. 2012 Jan; 34(1): 93-106.
In article      View Article  PubMed
 
[4]  Jiang X, Andjelkovic AV, Zhu L, Yang T, Bennett MVL, Chen J, Keep RF, Shi Y. Blood-brain barrier dysfunction and recovery after ischemic stroke. Prog Neurobiol. 2018 Apr-May; 163-164: 144-171.
In article      View Article  PubMed
 
[5]  Gorman AM. Neuronal cell death in neurodegenerative diseases: recurring themes around protein handling. J Cell Mol Med. 2008 Dec; 12(6A): 2263-80.
In article      View Article  PubMed
 
[6]  Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012 Apr; 33(7): 829-37, 837a-837d.
In article      View Article  PubMed
 
[7]  Shalini S, Kumar S. Caspase-2 and the oxidative stress response. Mol Cell Oncol. 2015 Jan 23; 2(4): e1004956.
In article      View Article  PubMed
 
[8]  Doré S. Unique properties of polyphenol stilbenes in the brain: more than direct antioxidant actions; gene/protein regulatory activity. Neurosignals. 2005; 14(1-2): 61-70.
In article      View Article  PubMed
 
[9]  Sritularak B, Anuwat M, Likhitwitayawuid K. A new phenanthrenequinone from Dendrobium draconis. J Asian Nat Prod Res. 2011 Mar; 13(3): 251-255.
In article      View Article  PubMed
 
[10]  Treml J, Leláková V, Šmejkal K, Paulíčková T, Labuda Š, Granica S, Havlík J, Jankovská D, Padrtová T, Hošek J. Antioxidant Activity of Selected Stilbenoid Derivatives in a Cellular Model System. Biomolecules. 2019 Sep 9; 9(9): 468.
In article      View Article  PubMed
 
[11]  Flefel EM, El-Sofany WI, Al-Harbi RAK, El-Shahat M. Development of a Novel Series of Anticancer and Antidiabetic: Spirothiazolidines Analogs. Molecules. 2019 Jul 9; 24(13): 2511.
In article      View Article  PubMed
 
[12]  Pinkhien T, Petpiroon N, Sritularak B, Chanvorachote P. Batatasin III Inhibits Migration of Human Lung Cancer Cells by Suppressing Epithelial to Mesenchymal Transition and FAK-AKT Signals. Anticancer Res. 2017 Nov; 37(11): 6281-6289.
In article      View Article
 
[13]  Desai SJ, Prickril B, Rasooly A. Mechanisms of Phytonutrient Modulation of Cyclooxygenase-2 (COX-2) and Inflammation Related to Cancer. Nutr Cancer. 2018 Apr; 70(3): 350-375.
In article      View Article  PubMed
 
[14]  Czupalla CJ, Yousef H, Wyss-Coray T, Butcher EC. Collagenase-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry. Bio Protoc. 2018 Nov 20; 8(22): e3092.
In article      View Article
 
[15]  Verma A, Verma M, Singh A. Animal tissue culture principles and applications. Animal Biotechnology. 2020: 269-93.
In article      View Article  PubMed
 
[16]  Karakaş D, Ari F, Ulukaya E. The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts. Turk J Biol. 2017 Dec 18; 41(6): 919-925.
In article      View Article  PubMed
 
[17]  Kyrylkova K, Kyryachenko S, Leid M, Kioussi C. Detection of apoptosis by TUNEL assay. Methods Mol Biol. 2012; 887: 41-7.
In article      View Article  PubMed
 
[18]  Daneman R, Prat A. The blood-brain barrier. Cold Spring Harb Perspect Biol. 2015 Jan 5; 7(1): a020412.
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