Aldina, Rosy and Prof.dr. M. Aris Widodo,, MS.,SpFK.,Ph.D. and dr. Hidayat Sujuti,, M.Sc.,Ph.D.,SpM. and Dr.drg. Nur Permatasari,, M.Si. (2018) Pengaruh Genistein Topikal Terhadap Sindroma Dry Eye Pada Tikus Ovariektomi Melalui Peran Sel Epitel Dan Goblet Konjungtiva Serta Sel Punca Epitel Limbal. Doktor thesis, Universitas Brawijaya.
Abstract
Dry Eye Syndrome (DES) is a multifactorial disease with a damaging potential to the ocular surface and tear film. The ocular surface is a specific mucous that is located in the conjunctiva, corneoscleral limbus, and cornea. Hiperosmolarity is a central mechanism, resulting in inflammation, damage and symptom on ocular surface. The main prevalence of DES is in post menstrual woman, resulting in the decreased estrogen excretion (particularly 17-estradiol) and causes complex Receptor Estrogen (RE)-ligan in nucleus to decrease. It is known that RE is in conjuctival epithel which is estrogen-sensitive. Also, RE function is to regulate stemness or stem cell differentiation. Limbal is progenitor cell having responses to homeostasis, a main source of regeneration as well as barriers of ephitelial cell of cornea and conjunctival epithelial cells. Genistein can substitute 17-estradiol as ligan bound with RE, despite its lower affinity compared to estrogen. Complex RE-genistein will inhibit promotor activation of proinflammatory cytokine gene, so that expression of proinflamatory cytokine will decline and inflammatory process on cornea will also be declined. Goblet cell apoptosis process declines, resulting in the increased mucin production. The decreased inflammation and the increased mucin production are expected to improve ocular surface. Some literatures stated that flavonoid as inflammatory agent does not have definitive mechanism or single so that it all explains that all effects of phenol have specific signals, depending on cell type. Up to now, the role of estrogen hormone on etio-pathomechanism of DES and inflammation of lacrimal gland has still been subject of debates, inconclusive and controversial. Thus, this research aims to find out the the impact of genistein on SDE, through the role of REβ, NF-kB, IL-1β conjunctiva epithelial cell; MUC5AC of conjuctival goblet cell;; p63, K12, and K13 limbal epithelial stem cell on SDE mouse cell with ovariectomy. This is pure experimental research in vivo. After the mouse experiences 1 month of ovariectomy, DES diagnostic test is performed, consisting of Schirmer test, FBUT, and Ferning. DES is given topical genistein 4 times a day over a week. Each groups is given dosages of 50 M, 100 M, 200 M; while the control group is given BSS. The preparation of tissue sample is done through paraffin block. Next, is the excision phase. The proper excision will result a ribbon-shaped section. After preparation, the specific primary antibody given is anti rat RE antibody; anti rat IL-1 antibody; anti rat NF-kB antibody; anti rat MUC5AC antibody; anti rat K12 antibody; anti rat K13 antibody. Having done that, the secondary antibody incubation that is given the FITC label for IL-1, MUC-5AC, and K12 will give off a green luminescence. Rhodamin label for RE, NF-kB, K13 will give off reddish luminescence. Meanwhile, p63 with the secondary anti mouse IgG antibody that is labeled with peroxidase will give off brownish luminescence. Examination is done through Olympus fluo FV 10-ASW 1.7type, LSCM confocal laser microscope with 400 times magnification with 5 fields of view. According to the data analysis, there has been a significant increase of RE- on the 100 M, but still below the negative control group. Average comparison of 100 μM genistein RE- differs from 50 μM and 200 μM genistein RE-, positive and negative control, indicating the presence of RE- regulation – ligan on the conjunctival epithelial cell. It is shown that the average expression ratio of IL-1 is the lowest in the 100 μM treatment group and IL-1 increases in the positive control instead of the negative control. There is a different NF-kB expression on the genistein group with a significant value. The 100 μM treatment group can more easily decrease NF-kB than the 50 μM dan 200 μM treatment. 50 μM genistein treatment decrease NF-kB much more easily than the 200 μM. The 200 μM genistein treatment is still better at decreasing NF-kB than the positive control, which means that genistein inhibits the transcription factor of NF-kB that plays an important role in conjunctival epithelial cell inflammation on DES. The highest average expression ratio of MUC-5AC belongs to the 100 M genistein treatment. There seems to be a significant expression difference of MUC-5AC on the genistein treatment groups. On the positive control group, the MUC-5AC expression result decreases in comparison to the negative control group, which can only means that the conjunctival goblet cells on the SDE experience a decrease of expression in MUC-5AC. Meanwhile, on the limbal epithelial stem cell, there has been a difference in the K12 expression on the genistein treatment groups. 200 μM genistein treatment group can decrease K12 more, than the 100 μM treatment. On the limbal epithelial stem cell, there have been some K13 expression differences on the genistein treatment group. 200 μM genistein treatment group can decrease K13 more easily than 100 μM dan 50 μM treatment. On the limbal epithelial stem cell, there have been some distinct differences in the p63 expression on the genistein treatment groups. The average p63 expression on the positive control group differs from the 100 μM and 200 μM treatment, and the negative control. P63 on the positive control group has no significant difference in comparison with the 50 μM dosage. The result of statistical analysis using path analysis with Partial Least Square approach for IL-1 toward DES, t-statistics 2.128, this indicates that there is a significant, direct effect between IL-1 and DES making up 0.264. The positive effect between IL-1 on DES It means that the increase of IL-1 causes the increase of DES. It also goes both ways, the decrease of IL-1 causes the decrease of DES. IL-1 towards K12 shows a t-statistics of 3.011, indicating that there is a significant, direct impact of IL-1 towards K12 equal to 0,614. The positive effect between IL-1 on K12 it means that it can be concluded that the increase of IL-1 causes the increase of K12. It also goes both ways, the decrease of IL-1 causes the decrease of K12. The direct impact of IL-1 towards K13 is equal to 0.974 with a t-statistics of 6,434, indicating that there is a significant. The positive effect between IL-1 on K13 it means that it can be concluded that the increase of IL-1 causes the increase of K13. It also goes both ways, the decrease of IL-1 causes the decrease of K13.The direct impact of IL-1 towards MUC5AC is equal to -0.732 with t-statistics of 9.655, indicating that there is a significant. The negative effect between IL-1 on MUC5AC it means that it can be concluded that the increase of IL-1 causes the decrease of MUC5AC. It also goes both ways, the decrease of IL-1 causes the increase of MUC5AC.The direct impact of IL-1 towards p63 results in the t-statistics of 2.447, indicating that there is a significant, direct impact that amounts to-0.560. The negative effect between IL-1 on p63 it means that it can be concluded that the increase of IL-1 causes the decrease of p63. It also goes both way, the decrease of IL-1 causes the increase of p63. K13 towards DES shows a t-statistics of 8.258, indicating that there is a significant, direct impact of K13 towards DES equal to 0.648. The positive effect between K13 on DES it means that it can be concluded that the increase of K13 causes the increase of DES. It also goes both ways, the decrease of K13 causes the decrease of DES. K12 towards K13 shows a t-statistics of 3.031, indicating that there is a significant, direct impact of K12 towards K13 equal to 0.302 . The positive effect between K12 on K13 it means that it can be concluded that the increase of K12 causes the increase of K13. It also goes both ways, the decrease of K12 causes the decrease of K13. MUC5AC towards DES shows a t-statistics of 3.515, indicating that there is a significant, direct impact of MUC5AC towards DES equal to -0.372. The negative effect between MUC5AC on DES it means that it can be concluded that the increase of MUC5AC causes the decrease of DES. It also goes both ways, the decrease of MUC5AC causes the increase of DES. NF-kB towards DES shows a t-statistics of 2.344, indicating that there is a significant, direct impact of NF-kB towards DES equal to -0.315. The negative effect between NF-kB on DES it means that it can be concluded that the increase of NF-kB causes the decrease of DES. It also goes both ways, the decrease of NF-kB causes the increase of DES. NF-kB towards IL-1 shows a t-statistics of 4.935, indicating that there is a significant, direct impact of NF-kB towards IL-1 equal to 0.682. The positive effect between NF-kB on IL-1 it means that it can be concluded that the increase of NF-kB causes the increase of IL-1. It also goes both ways, the decrease of NF-kB causes the decrease of IL-1. RE- towards IL-1 shows a t-statistics of 2.023, indicating that there is a significant, direct impact of RE- towards IL-1 equal to -0.260. The negative effect between RE- on IL-1 it means that it can be concluded that the increase of causes the decrease of IL-1. It also goes both ways, the decrease of RE- causes the decrease of IL-1. RE- towards K12 shows a t-statistics of 0.190. It is no direct effect significant between RE- towards K12 with a weak coefficient path of 0, 031. In other words, high low RE- does not effect to increase or decrease of K12. RE- towards K13 shows a t-statistics of 4.062, indicating that there is a significant, direct impact of RE- towards K13 equal to 0.596. The positive effect between RE- on K13 it means that it can be concluded that the increase of RE- causes the increase of K13. It also goes both ways, the decrease of RE- causes the decrease of K13. RE- towards NF-kB shows a t-statistics of 24.466, indicating that there is a significant, direct impact of RE- towards NF-kB equal to -0.836. The negative effect between RE- on NF-kB it means that it can be concluded that the increase of RE- causes the decrease of NF-kB. It also goes both ways, the decrease of RE- causes the increase of NF-kB. RE- towards p63 shows -0.144 with a t-statistics of 0.518, It is no direct effect significant between RE- towards p63. In other words, high low RE- does not effect to increase or decrease of p63. It is concluded that dosage of 100 μM of genistein increases expression of RE-, MUC-5AC with the highest mean; and expression average of NF-kB, IL-1 the lowest. Genistein of 200 μM dose can decrease K12, K13 more than 100 μM and 50 μM doses. Genistein of 200 μM can increase expression of p63 having the highest average. There is significance direct effect between genistein dose towards RE-; IL-1 towards DES; between IL-1 and K12; between IL-1 and K13; between between IL-1 and MUC5AC; between IL-1 and p63; between K13 and DES; between K12 and K13; between MUC5AC and DES; NF-kB and DES; NF-kB and IL-1; between RE- and IL-1; RE- and K13; RE- and NF-kB. There is no significant direct effect between RE- and K12; between RE- and p63.
| Item Type: | Thesis (Doktor) |
|---|---|
| Identification Number: | DIS/617.7/ALD/p/2018/061901510 |
| Subjects: | 600 Technology (Applied sciences) > 617 Surgery, regional medicine, dentistry, ophthalmology, otology, audiology > 617.7 Ophthalmology |
| Divisions: | S2/S3 > Doktor Ilmu Kedokteran, Fakultas Kedokteran |
| Depositing User: | soegeng sugeng |
| Date Deposited: | 10 Oct 2022 08:39 |
| Last Modified: | 10 Oct 2022 08:39 |
| URI: | http://repository.ub.ac.id/id/eprint/195493 |
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