2007 Research Report From Dr.Levin and Dr.Lin, Albany, New York

Protection against ischemia and ischemia followed by reperfusion by Antrodia Camphorata


Final Report


Robert M. Levin Ph.D. and Alpha D. Lin M.D.

Albany College of Pharmacy and Stratton VA Medical Center

Urology Division, National Yang-Ming University, and Taichung Poah-Ai hospital Taiwan

Taiwan Well Shine Biotech Company, Taiwan






Introduction
There is growing evidence that ischemia (reduced blood flow to an organ system) and reperfusion following ischemia (the generation of free radicals and subsequent oxidative damage to tissues and organs) are directly involved in several degenerative diseases associated with ageing including heart disease, kidney dysfunction, liver dysfunction, CNS degeneration, and urinary bladder dysfunction (1-8). In general antioxidant therapies have proven to be effective in both reducing the progression of ischemia / reperfusion injury, and in the treatment of ischemic / reperfusion damage (9-16). The major targets for ischemic / reperfusion injury are mitochondria, sarcoplasmic reticulum, and nerves (1,7,8,11,14-16).
We have found the urinary bladder to be an excellent model organ system to study ischemia / reperfusion injury (1,14-18). Urinary bladder dysfunction is a major affliction of aging men (19-21); more than 80% of men over the age of 50 require medical attention for bladder dysfunction. In men, it is a progressive disease that results from a slowly increasing prostate mass compressing the urethra, eventually causing the characteristic bladder symptoms of urgency, frequency and poor urine flow (19-21). As the prostate gradually enlarges, bladder function can remain relatively "normal" for many years as the organ compensates for the progressive increase in urethral resistance through hypertrophy (an increase in bladder wall thickness and net increase in bladder mass) (22,23). It is not until the bladder shifts to decompensated function that moderate to severe alterations occur and the patient seeks medical attention.
The progression of obstructed bladder dysfunction is related directly to the the increased wall thickness resulting in ischemia / reperfusion injury characterized by damage to nerves, synapses and smooth muscle cells within the bladder wall, and due, in part, to generation of reactive oxygen and nitrogen species that functionally damage plasma and subcellular membranes (24,25).
It is clear from published studies that Antrodia camphorate has significant cell-protective properties (26-29), which would make it an excellent candidate for the prevention and / or treatment of ischemic / reperfusion damage
In conclusion, we believe that if we reduce the level of ischemia / reperfusion, we would significantly reduce the progression of the pathogenesis of ischemic bladder dysfunction described above. From the studies in the literature, Antrodia camphorate extracts may be very effective in the treatment of ischemic bladder dysfunction. On a broader scale, the demonstration that Antrodia camphorate protects the bladder against ischemia / reperfusion injury would indicate that this agent would probably be very effective in the protection and treatment of other organs against ischemia / reperfusion damage; ie congestive heart failure, alzheimers, kidney disease, etc.

Specific Aims
The specific aim of our proposal is to show that orally administered Antrodia camphorata can protect rabbit bladders from the progressive dysfunctions induced by experimental bilateral ischemia / reperfusion. The preparation will be administered daily by gastric lavage to insure that uniform amounts of extracts are ingested.

Experimental Design
Antrodia camphoata (AC) preparation:
Each pellet of AC manufactured by Taiwan Well Shine Biotech Company contains 250 mg powder. For man, the daily dose is 6 capsules / day = 1500 mg/day. For a 4 kg rabbit, the dose would be 100 mg / day suspended in 10 ml room temperature water and given to the rabbit daily by oral gavage.
Specifically, we have removed the powder from the commercial capsules, weighed out the proper amount and stirred the powder in room temperature water slowly so we did not destroy the cell wall of AC.
Animal Procedures: Thirty six male New Zealand White rabbits (3-4 kg) were separated into 6 groups of 6 animals each. Rabbits in groups 1 - 3 were fed 100 mg Antrodia camphoata suspensions daily as described above; those in groups 4 - 6 received vehicle. All rabbits received their respective treatments daily for 3 weeks.
After 3 weeks, all rabbits were anesthetized and cystometries performed. After cystometry each rabbit in groups 1,2,4,and 5 were subjected to bilateral ischemia, created by placing mini vascular clamps on both vesical arteries for a period of 2 hours. At the end of the two hours, the clamps were removed and the rabbits in groups 1 and 4 allowed to recover for 2 hours in order to recover the cellular levels of ATP; the rabbits in groups 2 and 5 were allowed to recover for one week (reperfusion period). The rabbits in groups 3 and 6 were sham operated and allowed to recover for 1 week. Feeding was continue during the recovery period.
Upon completion of the experiment, each rabbit was anesthetized and cystometry repeated. Immediately after cystometry the bladder was excised rapidly and weighed.
Isolated muscle strip pharmacology: Then, three isolated strips from each bladder were mounted in separate baths and the contractile responses to FS, carbachol, ATP and KCl were determined by standard methods.
Biochemistry: The balance of each bladder was separated into its muscle and mucosal compartments, frozen in liquid nitrogen and stored at -70oC for biochemical analyses. We measured citrate synthase (CS) activity as a marker for mitochondrial function, SR Ca+2-ATPase (SERCA) as a marker for SR function and choline acetyltransferase (ChAT) activity as a marker for cholinergic nerve function.

These studies will answer the following questions:

1. Does Antrodia camphorate protect the urinary bladder from ischemia and ischemia followed by reperfusion?

2. Does Antrodia camphorate protect bladder nerves from ischemia and ischemia followed by reperfusion?

3. Does Antrodia camphorate protect detrusor muscle mitochondrial and SR membranes from ischemia and ischemia followed by reperfusion?

Positive results from these studies would strongly support the usefulness of Antrodia camphorate for the prevention of progressive lower urinary tract dysfunction in the aging male population.




Progress Report:

Figure 1 At this time, all physiological studies have been completed. The ischemia group followed by 2 hours recovery showed no positive (or negative) effects of Antrodia, and have been eliminated from the report and subsequent manuscript. Ischemia / Reperfusion in the following figures represent ischemia followed by 1 week reperfusion.

Figure 1 shows that ischemia reperfusion (I/R) results in a mild but significant increase in bladder weight. No significant increase in bladder weight was noted for the I/R Antrodia group.


B
A
Figure 2 Figure 2 shows the responses to field stimulation. Panel A displays the maximal contractile response while panel B displays the maximal rate of tension generation. For both parameters, Antrodia mediated a significant increase in the response in the control bladders to all frequencies of stimulation. I/R resulted in significant decreases in the responses to all frequencies of stimulation. In the presence of Antrodia, the responses to all frequencies of stimulation were significantly greater than the responses in the absence of Antrodia.
Figure 3Figure 3 displays the response to ATP, carbachol, and KCl. Antrodia resulted in a significant increase in the responses to all forms of stimulation in the control rabbits. I/R resulted in significant decreases in the responses to all forms stimulation; although the magnitude of the decrease was less than the magnitude of the decreased responses to FS. Antrodia resulted in completely inhibiting the reduced contractile responses to ATP and KCl, and a significantly reduced decrease in the magnitude of the decreased response to carbachol.





Figure 4 AFigure 4 shows the cystometric curves. For both control and I/R, the cystometric curves for the Antrodia treated rabbits were significantly below those of the no treatment group. This indicates that compliance of the control and the I/R bladders were significantly reduced under both conditions by Antrodia.

Figure 4 B













Figure 5Figure 5 shows the compliance as intravesical pressure change over the first 20% capacity. I/R resulted in a significant decrease in compliance indicating a stiffer bladder. Antrodia treatment resulted in a significantly more compliant bladder under both control and I/R conditions. In regard to the IR bladder, there was no change in compliance for the Antrodia treated bladder.



Figure 6Figure 6 shows the micturition pressure and the volume at micturition derived from the cystometry studies. Antrodia treatment had no effect on either parameter for the control bladders. I/R resulted in no change in micturition pressure and a significant decrease in the volume at micturition. Antrodia treatment of the I/R bladders showed a significantly decreased volume at micturition compared to the no treatment groups.

Figure 7 shows the effect pf Amtrodia on citrate synthase activity. I/R mediated a significant decrease in citrate synthase activity of the mucosa. No change was noted in the activity of the smooth muscle. The rabbits treated with Antrodia showed a significantly higher citrate synthase activity of the mucosa after I/R when compared to the activity of the rabbits receiving vehicle.





Figure 8 shows that I/R resulted in a significant decrease in the choline acetyltransferase (Chat) activity of both treatment groups. Antrodia had no significant effect on the Chat activity of control or I/R groups.







Figure 9 shows the calcium ATPase activity. I/R had no significant effect on the total clcium ATPase activity; nor did treatment with Antrodia.










Figure 10 shows the SERCA activity (thapsigargin-sensitive calcium ATPase activity). I/R had no effect on SERCA activity of muscle, however, I?R resulted in an increased activity of the mucosa. Antrodia treatment resulted in a significant increase in the SERCA activity of the muscle but no effect on the activity of the mucosa.







Discussion and Conclusions:

One of the most striking results of this study is the demonstration that orally feeding the rabbits Antrodia Camphorata improves urinary bladder function in the control animals. The bladders have increased contractile responses to all forms of stimulation tested, and improved bladder compliance. At this point we do not have a mechanism of action for the increased contractile responses nor for the increased compliance. The biochemical studies have demonstrated that Antrodia had no effect on citrate synthase activity, choline acetyltransferase activity, total calcium ATPase activity, nor SERCA activity. Future studies will look at the smooth muscle collagen ratio. It is possible that Antrodia may result in an improved smooth muscle to collagen ratio in the bladder body which may be involved in the beneficial effects observed in the control condition.

I/R mediated the following changes in the no treatment group: increased bladder weight, decreased contractile responses to all forms of stimulation, decreased compliance (stiffer bladder), and decreased volume at micturition. In addition, I/R mediated a decreased activity for citrate synthase and Chat; but no change in total calcium ATPase activity. Antrodia treatment of the I/R bladders resulted in the following: decreased bladder weight back to control values, increased contractile responses to all forms of stimulation; the responses to ATP and KCl returning to control values while the responses to FS and carbachol were still significantly below control levels; increased (improved) compliance and decreased volume at micturition. In addition, Antrodia treatment resulted in an increased citrate synthase activity, although the activity did not get back to control levels. Antrodia had no effect on choline acetyltransferase activity or on total calcium ATPase activity of the I/R bladders; however, Antrodia resulted in a significant increase in the SERCA activity of the smooth muscle above control activity. There was no effect of Antrodia on the SERCA activity of the mucosa. The increased SERCA activity of the smooth muscle is consistent with the increased contractile responses; since the mucosa does not have a contractile function, it was not supriosing there was no effect on mucosal SERCA activity.
These all are beneficial effects which demonstrate that Antrodia treatment both improved normal bladder function, and protected the bladder against the detrimental changes mediated by ischemian / reperfusion.
This completes the studies; however, we are continuing with additional studies to try to determine the mechanisms by which Antrodia has it’s beneficial action. These additional studies include histological studies. No additional funds are requested.

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