TUDCA Protects Retinal Pigment Epithelial cells from oxidative Injury and Endoplasmic Reticulum Stress In Vitro. For Scientific Free Lectures, Visit - http://bit.ly/VisitZofirAcademy

1. Presenting By
Arman Firoz
20PhD0047
Tauroursodeoxycholic Acid Protects Retinal
Pigment Epithelial Cells from Oxidative Injury and
Endoplasmic Reticulum Stress In Vitro
ARTIC LE
REEM HASABALLAH ALHASANI 1,2 , MOHAMMAD ALMARHOUN 2, XINZHI ZHOU 2, JAMES REILLY 2, STEVEN PATTERSON 2, ZHIHONG
ZENG 3,* AND XINHUA SHU 2,4,5,*
Guide
Dr.Priti Talwar
1

2. Visual processing in the human
2

3. https://www.youtube.com/watch?v=vvrlpVPxYiE
Reduced Central Vision
What are the symptoms?
Yellowish Fleck
Reduced Colour Perception
What happens when the system is dysregulated !!!
3

4. TUDCA RESCUES RPE FROM UNDERGOING
APOPTOSIS
4

5. Taurine-conjugated bile acid
(low level in human bile)
• TUDCA stands for Tauroursodeoxycholic acid
• It is a biological substrate which is naturally made by human body
TAURINE + URSODEOXYCHOLIC ACID = TUDCA
BILE
(UDCA = URSODEOXYCHOLIC ACID)
Benefits of TUDCA
What is TUDCA
How exactly is TUDCA formed in Human Body ?
Huntington's disease
Parkinson's disease
Stroke
Retinal degenerative disorders
Early cell death
Prevents
5

6. RETINAL DEGENERATIVE DISORDERS
Protective effect against photoreceptor
degeneration in Rpgr knockout mouse
model of retinitis pigmentosa.
DOI: 10.1002/jcp.28519
Protective role of TUDCA against
retinal degeneration in retinal cell
lines and animal models
Pubmed: 25443293
TUDCA treatment ameliorates
photoreceptor death
http://www.molvis.org/molvis/v12/a195/
Protection of ganglion cells from
n-methyl-d-aspartate-induced
retinal damage in rats
https://doi.org/10.1371/journal.pone.0137826
Protection of ganglion cells from
crushed optic nerve
https://doi.org/10.1038/s41598-018-36473-2
6

7. In this study, they have evaluated the PROTECTIVE POTENTIAL of TUDCA against oxidative
damage and endoplasmic reticulum (ER) stress in human RPE cells. They found that TUDCA
ATTENUATED H2O2-evoked oxidative damage and inhibited thapsigargin (TG)-induced ER stress.
7
WHAT CAUSES RETINAL CELL DEATH ?
Sustained ER stress trigger an inflammatory response and exacerbate oxidative stress,
both of which contribute synergistically to tissue damage.
The UPR up-regulates ER chaperones, reduces protein translation, and promotes clearance of
cytotoxic misfolded proteins to restore ER homeostasis. If this vital process fails, the cell will be
signaled to enter apoptosis, resulting in cell death.

8. Materials and Methods
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
8

9. 9
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
- ARPE-19 cells were cultured in with DMEM/F12
- 5 x 104 cells/well were seeded in 96 well plate for the Treatment  Kept for 24 hours incubation
H2O2 TG TUDCA H2O2+TUDCA TG+TUDCA
MTT Assay was performed to analyse the cell viability
For 24 Hours

10. 10
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
PBS wash  Fixed in Formaldehyde  PBS Wash  Permeabilization 0.2% Triton X 100
rTDT reaction mix incubation  saline-sodium citrate (SSC) buffer
Cells were mounted with DAPI  Quantification of TUNEL positive cells

11. 11
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
DCFH-DA was used to detect total ROS
- ARPE-19 cells were cultured in with DMEM/F12
- 5 x 104 cells/well were seeded in 96 well plate for the Treatment  Kept for 24 hours incubation
H2O2 H2O2+TUDCA
For 24 Hours

12. 12
12
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
- ARPE-19 cells were cultured in with DMEM/F12
- 5 x 105 cells/well were seeded in 6 well plate for the Treatment  Kept for 24 hours incubation
H2O2 H2O2+TUDCA
RNA extracted using TRIZOL  cDNA  qPCR on targeted genes
For 24 Hours

13. 13
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
Caspase-3 and -7 activities were measured with a Caspase-Glo 3/7 assay kit (Cat. G8090, Promega,
Southampton, UK) following the manufacturer’s guidelines.

14. 14
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
- ARPE-19 cells were cultured in with DMEM/F12
- 5 x 105 cells/well were seeded in 6 well plate for the Treatment  Kept for 24 hours incubation
H2O2 H2O2+TUDCA
Culture Media was Collected followed by analysis of IL-6 and TNFa expression
For 24 Hours

15. 15
1. Cell Viability
2. Detection of Apoptosis
3. Quantification of Reactive Oxygen Species (ROS) Production
4. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
5. Measurement of Caspase-3/7 Activities
6. Enzyme-linked Immunosorbent Assay (ELISA)
7. Statistical Analysis
GraphPad Prism 6 software

16. 1. Effects of H2O2 and TUDCA on Cell Viability
2. TUDCA Attenuated H2O2-induced Oxidative Stress in RPE Cells
3. TUDCA Inhibited H2O2-Induced Expression of Proinflammatory Cytokines in RPE Cells
4. TUDCA Attenuated Thapsigargin-Induced ER Stress in RPE Cells
16
Results

17. Effects of H2O2 and TUDCA on Cell Viability
TUDCA rescued the cells from undergoing apoptosis
17
Figure 1

18. TUDCA decreases oxidative stress-induced caspase 3 expression and caspase 3/7 activity in ARPE-19
cells.
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Effect of TUDCA on Caspase 3 and 7
Caspase-3 and -7 activities were measured with a Caspase-Glo 3/7 assay kit (Cat. G8090, Promega, Southampton, UK) following the manufacturer’s guidelines.
Figure 2

19. TUDCA suppresses the oxidative stress in the presence of H2O2 at mRNA level
19
reduction of cell damage. Protects cells from oxidative stress
defense against oxidative stress reduces apoptosis defense against oxidant aggression
Figure 3

20. TUDCA suppresses the oxidative stress in the presence of H2O2 at Protein level
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Oxidative stress marker
Figure 4

21. TUDCA suppressed H2O2-induced inflammation.
21
Figure 5
qRT-PCR
ELISA

22. TUDCA counteracted the effects of thapsigargin (TG, 1μM) on ARPE-19 cell viability and apoptotic cell death by
suppressing ER stress
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Figure 6 Figure 7

23. DISCUSSION
8·7% of the worldwide population has age-related macular degeneration, and the projected number of people with
the disease is around 196 million in 2020, increasing to 288 million in 2040.
Oxidative and ER stress plays a critical role in the progression of inherited and complex retinal Degeneration.
Suppression of oxidative and ER stress represents a promising therapeutic strategy for the treatment of retinal degeneration.
Current study demonstrated that TUDCA attenuated cell death, decreased ROS production, upregulated
antioxidant gene expression, and inhibited inflammation in H2O2-treated RPE cells. TUDCA
treatment also suppressed TG-induced ER stress and associated cell death.
TUDCA has been demonstrated to have anti-inflammatory activity in the retinas of streptozotocin-induced diabetic rats.
TUDCA alleviated H2O2-induced inflammation in RPE cells, decreasing expression and secretion of proinflammatory cytokines, IL-1, IL-6,
and TNF-a
CONCLUSION
This work demonstrates that TUDCA can protect RPEfrom oxidative stress, inflammation, and ER stress, and further supports
that TUDCA has therapeutic potential for treating retinal degeneration.

24. • Bright field image of cells under treatment would have clarified many assumptions
• Experiments could have done chronic way also.
• Pre or post treatment could have included to see the exact rescuing effect.
• Could have included primary RPE cells for further validation.
• TUNEL Assay for TUDCA alone could have added in the first session
• RPE cell Functionality study also could have included (E.g. Phagocytosis)
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Critique

25. THANK YOU