Difference between revisions of "Team:TAS Taipei/Experimental Summary"

Through literature research, we found that glutathione (GSH) and 25-hydroxycholesterol (25HC) should prevent and treat cataracts, respectively. We wanted to first see if we could simulate cataract formation and reproduce the effects of GSH and 25HC seen by other researchers. If successful, then we can later test our own constructs using the same cataract model.

We set up the model by extracting soluble proteins from the lens of Priacanthus macracanthus, a common freshwater fish that we purchased from a market (Figure 01 left). There are two distinct parts to the lens: an outer soft layer called the cortex and an inner layer called the nucleus (Figure 01 right). We focused on the lens nucleus, because that part contains older cells and is more prone to cataract formation. The lens nucleus was placed into Tris Buffer and gently shaken overnight. After centrifuging, the supernatant contains dissolved protein from the lens nucleus. We then incubated the lens solution with hydrogen peroxide (H2O2), since H2O2 is the main reactive oxygen species that oxidizes lens proteins and induces cataracts.

To quantify the severity of cataracts in our model, we used spectrophotometer to measure absorbance of the protein solution (Figure 03). As lens proteins get oxidized, absorbance should increase because protein clumps that form will scatter the emitted light. To find a wavelength of light for data collection, we compared the absorbance of an untreated proteins, H2O2-treated proteins, and heat-denatured proteins as a positive control. We observed an absorbance peak at 397.5 nm for insoluble lens proteins, and chose to collect all future absorbance values at that wavelength.

We tested different concentrations of H2O2 on this protein solution to see if cataracts form. Our results show that increasing concentrations of H2O2 lead to more severe cataracts (Figure 04). We also ran a protein gel to compare the sizes of untreated and H2O2-treated proteins. After treatment with H2O2, there was an increase in higher bands, which is consistent with the idea that proteins are clumping and aggregating (Figure 05). Together, the protein gel and our lens cataract model suggest that our model accurately represents cataract development.

GSH is the main antioxidant in the lens and prevents H2O2 from oxidizing crystallin proteins. After GSH converts H2O2 into water, it becomes GSSG, which will be recycled back to GSH with the help of glutathione reductase (GSR) (figure 06). Older cells in the nucleus are unable to produce GSR efficiently, so over time GSSG builds up. Our project is to deliver the GSR into the lens in order to facilitate the conversion of GSSG to GSH. We purchased GSH from Sigma Aldrich. 8 mg of GSH was added to the protein solution prior to the addition of H2O2 .

Protein solutions with GSH and H2O2 have an absorbance value lesser than those with just protein solutions with H2O2 . The smaller absorbance value indicates smaller protein aggregation and shows GSH has preventative effect. (figure #)

We select 25 HC (25 hydroxycholesterol) to be our treatment because it reverses aggregation and restores solubility of the lens crystallin protein by stabilizing the natural state of the proteins. In order to verify our research, we use commercially bought 25 HC from Sigma Aldrich to treat the cataracts model. Commercial 25HC came in powder form, and was dissolved in 95% ethanol; thus, for our negative control, we added both H2O2 and ethanol into the protein solution. We also bought vet eyedrops for cataracts from OcluVet to be the positive control (Figure 1). Figure 2 shows the setup of the experiment.

Our goal is to develop noninvasive, easy-to-use, and affordable eyedrops to prevent and treat cataracts. Patients who already have cataracts need to reverse protein damage. Through literature research, we found a molecule called 25-hydroxycholesterol (25HC) that can reverser protein aggregation.

Prevention

Through literature research, we found an enzyme that recycles oxidized glutathione (GSSG) into glutathione (GSH), which neutralizes H2O2 and prevents crystallin damage. This enzyme is called glutathione reductase (GR) (Ganea & Harding, 2006). Even though GR exists in the lens, its amount decreases with age (Michael, 2011). We produced GR for delivery into the lens and prevention of cataracts.

Treatment

We also found a molecule that can restore solubility of protein clumps and lens transparency. It is called 25-hydroxycholesterol (25HC) (Makley et al., 2015). 25HC can be produced from cholesterol, which is abundant in the lens, by the enzyme cholesterol 25-hydroxylase (CH25H). We produced CH25H for delivery into the lens and treatment of cataracts.