Regulation of Antioxidants in Cardiovascular disease: A Study of Protandim on Nrf2 Regulation

Summary

Cardiovascular disease (CVD) is the number one cause of death in the world today, according to the American college of Cardiology. Among the known risk factors for CVD include genetic factors, high blood pressure, high cholesterol, smoking and diets high in salt. On the cellular level a high salt diet can lead to increased oxidative stress,  having detrimental effects on endothelial function leading to high blood pressure, heart attack and stroke. Oxidative stress has been a major area of investigation, especially in master regulators of antioxidant response like Nuclear factor (erythroid-derived-2)- like 2 (Nrf2). Several drug trials have been conducted in attempts to affect Nrf2 regulation and upregulate antioxidant elements, including trials with the commercially available phytochemical mixture Protandim. The present study investigated the effects of dietary salt intake and Protandim in Sprague-Dawley (SD) rats and Nrf2 -/- (KO) rats. Rats were fed a low-salt diet (LS) (0.4% NaCl), high-salt diet (HS) (4.0% NaCl), or high-salt diet with Protandim treatment (HS + Pro). To assess changes in oxidative stress in the presence and absence of Nrf2 and Protandim - catalase, MnSOD and SOD activities were assessed via colorimetric assay. In SD rats, the catalase activity for Hs+pro was significantly increased compared to animals on low salt and high salt diet alone(p=0.053), whereas there no significant increase in catalase activity with any treatment of Nrf2 KO animals(p=0.235). With an addition of a high salt diet and Protandim, there is a downward trend of superoxide dismutase(SOD) activity(p=0.235). Manganese SOD activity is nearly significantly reduced in high salt diet and Protandim treatment compared to low salt diet alone(p=0.187). In KO animals total SOD activity was not significantly different between treatment groups(p=0.470). These finding indicate that the treatment with Protandim could ameliorate some of the oxidative stress response to high salt diet in a Nrf2 dependent/independent manner. This experiment shows that Protandim can be an effective way of treating oxidative stress and finding ways to treat CVD.

 

Question / Proposal

Cardiovascular disease (CVD) is the number one cause of death in the world today, among the known risk factors for CVD include genetic factors, high blood pressure, high cholesterol, smoking, and diets high in salt. On the cellular level a high salt diet can lead to increased oxidative stress, having detrimental effects on endothelial function leading to high blood pressure, heart attack and stroke.

Many people find that they have on the go lifestyles, do to their busy routines with work or family, we often find ourselves lacking the time to ensure we maintain a healthy diet, and to find a way to ameliorate the detrimental effects of a longterm high salt diet. In the past 20 years fast food consumption has gone up significantly leading to the increased consumption of foods high in salt.

Through my research I hope to find a solution and better understanding to endothelial dysfunction caused by diets high in salt, this is becoming more important as obesity and CVD related to unhealthy eating are at their all time high.

Question:
Can the phytochemical mixture Protandim up regulate Nrf2 response channels and ameliorate the effects of a high salt diet?

Over time diets high in salt are known to lead to high blood pressure, heart attack, and stroke. It can also lead to heart failure. There is also some evidence that too much salt can damage the heart, aorta, and kidneys. Since the number one cause of death in the world is CVD, recently there has been more emphasis on researching master regulators of antioxidant production such as Nrf2. Up regulation of these channels are known to lead to an increased anti-oxidant production to combat the stressors related to CVD. Protandim has been available on the market as a phytochemical mixture composed of five naturally occurring phytochemicals: Milk thistle (Silybum marianum) extract, bacopa (bacopa monnieri) extract, ashwagandha (withania somnifera) root, green tea (camellia sinensis) extract and turmeric (curcuma longa) extract. All of these ingredients are known to contain antioxidant components and promotes the function of key regulatory enzymes like catalase and super oxide dimutase.

Hypothesis
The Intake of HS diet will increase oxidative stress as compared to a LS diet.  The increase oxidative stress due to high salt diet could be ameliorated by supplemental intake of phytochemical mixture Protandim.


These results show that Protandim is an effective way of ameliorating the effects of a high salt diet and general endothelial dysfunction, it also shows that further research dealing with master regulators and factors that promote key regulatory enzymes can be beneficial in decreasing the morbidity and mortality of CVD.

Research

Research in this area is becoming more popular in the scienific community, especially that which is focusing on master regulators such at Nrf2, to see what elements can be added in a diet to improve general cardiovascular disease. There has also been a lot of research done in the area of foods high in antioxidants, such as turmeric and ashwaghanda and the reason behind such high cardiovascular disease reports. On top of all of this research, we have seen a craze in "superfoods", foods that health "experts" claim are benefitial to heart health and overall wellbeing.

This research has shaped my project because it helped me come up with an idea and a passion towards the treatment of heart health and cardiovascular disease. Cardiovascular disease and stroke runs in my family both on my mother's and father's side of the family. My family is orginially from India and we eat Indian food almost everyday, since a young age I was engouraged to consume turmeric, a root from India that is known to have many antioxidant properties. This sparked my interest in this field of research, I wanted to find out what all the fuss was about in all these "superfoods". 

After further research in this area, watching documentaries, reading scholarly articles, I came across a shocking fact: cardiovascular disease is the number one cause of death in the world. After reading about what causes endothelial dysfunction and learning about the various mechanisms our body has to protect us from free radicals, I was inspired to pursue my own research. I think that it is important that scientists channel their efforts in this area because it promotes a healthy lifestyle and can decrease the morbidity and mortality of these diseases.

 

 

Method / Testing and Redesign

50 to 100 ml of previously flash-frozen rat liver were homogenized in 250ul homogenized in the assay buffer, using the MP Biosciences FastPrep-24 bead homogenization system.

The resulting homogenate was centrifuged at 10,000 x g for 15 min at 4oC, and supernatant stored at -80oC until use.

Total protein concentration was determined with Detergent-Compatible Bradford Assay (DC Assay) versus albumin standards (2.0mg/ml to 0.125 mg/ml) diluted in the matching homogenizing buffer.

 The present experiment used Cayman Chemical Catalase (Cat. 707002) and SOD (Cat 706002) assay kits. Assays were performed and analyzed in doublet according to manufacturer protocols. The resulting activities were normalized to total protein concentration and to LS activity, with error propagated accordingly. Statistical significance was determined by One-Way ANOVA, with n=6 per group.


The present experiment used sprague-dawley(SD) rats and Nrf2 -/-(KO).


All animal were on a low-sat diet(LS)- (0.4% NaCl), high-salt diet(HS)-(4.0% NaCl) or high salt diet with Protandim(HS+Pro)

 

This project was conducted at the Medical College of Wisconsin at the Lombard Lab of Physiology

Results

In SD rats, the catalase activity for HS + Pro was significantly increased compared to animals on low salt and high salt diet alone (p=0.235), whereas there was no significant difference in catalase activity with any treatment of Nrf2 KO animals (p=0.235).

With addition of high salt diet and Protandim there is a downward trend of superoxide dismutase(SOD) activity (p=0.053).

 MnSOD activity is nearly significantly reduced in high salt diet and Protandim treatment compared to low salt diet alone (p=0.187).

In KO animals total SOD activity was not significantly different between treatment groups (p=0.470

 

Links to graphs:

 

Figure 1 -  Total Catalase activity in nmmol/min/ml with Nrf2KO rats and SD rats

https://lh3.googleusercontent.com/-notcFschdpY/XA2ygoj5jXI/AAAAAAAAAK4/AGNlGfeYm5w3v0G6OHiEuyYs0mKm7vBMQCE0YBhgL/w300/1.png

 

Figure 2 -   Total Superoxide dismutase activity in U/ml with SD rats and Nrf2KO rats

https://lh3.googleusercontent.com/-gunsvTs8SoY/XA2yubfW-WI/AAAAAAAAAK8/LOkw3KQ4JigkaPURWuI2TaX1gfPkHmchQCE0YBhgL/w300/2.png

Figure 3 - %LS Catalase Activity of SD rats and Nrf2 -/- rats

https://lh3.googleusercontent.com/-Mf7C0mRr9u4/XA2zwunB_CI/AAAAAAAAALM/aI1W2gnsddAchTMe1c0QujIgD_Nh0ygHQCLcBGAs/w300/3.png

Figure 4 -  Total SOD Activity % of low salt in SD rats and Nrf2KO rats

https://lh3.googleusercontent.com/-ucWKoBAkBdc/XA20rp1_mqI/AAAAAAAAALs/gtbayo5568wQcByvZmOBkG-_whT98TSswCLcBGAs/w300/4.png

Figure 5 -  MnSOD activity %LS in SD rats and Nrf2KO rats

https://lh3.googleusercontent.com/-rsyoYhzBcWc/XA22nT1kt6I/AAAAAAAAAL8/Ze06665a8sEE05XpigNUBKxKhze0m1trACLcBGAs/w300/5.png

Figure 6 -  Average MnSOD activity in U/ml with SD rats and Nrf2KO rats

https://lh3.googleusercontent.com/-re3uO_scv2s/XA228pipK3I/AAAAAAAAAMU/PhcrXtcp4ycnH-w6ZJEgNQ1XdjE4aD85ACLcBGAs/w300/6.png

 

 

 

 

Conclusion

These findings indicate that the treatment with Protandim could ameliorate some of the oxidative stress response to high salt diet in a Nrf2 dependent manner. This experiment shows that Protandim can be an effective way of treating oxidative stress, and finding ways to treat Cardiovascular disease. These experiments show that Nrf2 is upregulated with the use of Protandim. Nrf2 works as a transcription factor by up regulating Catalase and SOD. The results show that when testing for SOD activity, the levels of SOD were not significantly different between Nrf2 KO rats and SD rats, showing that in both Nrf2 KO and SD rats the total SOD activity was increased when the protandim was applied. The data showed that in the SD LS rats the hypothesis was not supported, where there was actually a downward trend in total MnSOD activity. But in the KO rats, the hypothesis was supported because the HS+pro was almost equal to the LS, dropping down from the HS SOD activity. When looking at the total SOD Activity the hypothesis was supported, because the total SOD activity was increased after the addition of protandim to a high salt diet. When looking at the total Catalase activity, the data shows that the effects of the high salt diet were also ameliorated.

These present experiments answered my question by showing that Protandim is an effective solution for improving and maintaining endothelial homeostasis.

These findings do support my hypthothesis and expected outcome. My hypothesis was: the Intake of HS diet will increase oxidative stress as compared to a LS diet.  The increase oxidative stress due to high salt diet could be ameliorated by supplemental intake of phytochemical mixture Protandim. After analyzing my results, it is clear that protandim does ameliorate the effects of a high salt diet and help maintain endothelial homeostasis.

I would consider not using catalase in my testing, because since catalase specifically is an enzyme that catalyzes the reduction of hydrogen peroxide, it might be to general of an enzyme for specifically testing for the upregulation of Nrf2. I feel that Super oxide dimutase was a factor test because, Superoxide dismutase is an enzyme that specifically catalyzes the dismutation of the superoxide radical into either ordinary molecular oxygen or hydrogen peroxide. This question of what enzymes to use could also be considered for further research into what regulatory enzymes best upregulate Nrf2 and other master regulators.

 

In conclusion, Protandim can be an effective way of treating oxidative stress and ameliorating the effects of a high salt diet in a Nrf2 dependent manner. Protandim can be useful for treating the effects of oxidative stress and maintaining endothelial homeostasis. Further research towards analyzing antioxidant fluctuation and identifying antioxidant elements that promote key regulatory factors could be beneficial for decreasing the mortality and morbidity of cardiovascular diseases.

About me

https://youtu.be/ZdGPQ-XQ5QQ

 

Health & Safety

For saftey guidlines, please contact:

Kaleigh Kozak
Research Technologist I, Lombard Lab
(414) 955-8719 | kakozak@mcw.edu
 

Bibliography, references, and acknowledgements

References

Weber DS, and Lombard JH. Am J Physiol, 278: H500-506 (2000).

Priestley, JRC et al. Microvascular Research 89:134-145 (2013).

Tzemos N., et al. Hypertension, 51: 1525-1530, (2008).

Hansen-Smith et al. Circ. Res 79:324-330,1996

Zhu, J. et. al.   J. Vascular Res. 44:382-390 (2007).

McEwen et al., Microcirculation 16:220-234 (2009).

Durand, M et al. Am. J. Hyperten. 26:739-747, 2013.

Durand, M et al. Am. J. Physiol. 299:H1024-H1033, 2010.

McEwen, S. et al. Am J Physiol . 297:H1296-H1303 (2009).

Papaiahgari, S. et al. J. Biol. Chem. 279:42302-42312, 2004.

Priestley, J.R. et al. Am. J. Physiol. 310:H478-H487, 2016.

 

 

I would like to thank my parents, for driving me to the Medical College of Wisconsin, even in horrible Wisconsin weather.

I greatly appreciate the all the mentors that helped me at the Medical College of Wisconsin, including Dr. Julian H. Lombard and Ms. Kaleigh Kozak who spent many hours helping me understand lab equipment use and for helping providing me with animal samples.

I would also like to thank all the staff and research technologists at the Lombard Lab of Physiology for being so kind and helpful.