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Lornoxicam suppresses recurrent herpetic stromal keratitis through down-regulation of nuclear factor-kappaB: an experimental study in mice.

Yin J, Huang Z, Xia Y, Ma F, Zhang LJ, Ma HH, Li Wang L - Mol. Vis. (2009)

Bottom Line: Lornoxicam treatment significantly decreased the incidence of recurrent HSK, attenuated the corneal opacity scores, and also effectively suppressed both NF-kappaB activation and TNF-alpha expression in biological analysis.Histopathology examination revealed a reduced immunostaining positive cell density for NF-kappaB in the cornea from lornoxicam-treated mice as well as a diminished inflammatory response.Lornoxicam exerts protective effects against HSK, presumably through the down-regulation of NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People's Republic of China.

ABSTRACT

Purpose: We designed the current study to determine the protective effects of lornoxicam, a cyclooxygenase (COX) inhibitor, on recurrent herpetic stromal keratitis (HSK) and the nuclear factor-kappaB (NF-kappaB)-mediated mechanism in mice.

Methods: A corneal latent herpes simplex virus-1 (HSV-1) infected mouse model was established. Six weeks later, Ultraviolet B (UVB) irradiation induced the recurrence. Corneal swabs were obtained and cultured with indicator cells to determine shedding of the virus. Lornoxicam was administered intraperitoneally daily, beginning one day before irradiation and lasting for seven days. Saline-treated and mock-infected control groups were also studied at the same time. Development of corneal inflammation and opacity was scored. Immunohistochemical staining and an electrophoretic mobility shift assay were performed to evaluate the effect of lornoxicam on NF-kappaB activation in the corneal tissues. The levels of tumor necrosis factor-alpha (TNF-alpha) in the cornea were determined by an enzyme-linked immunosorbent assay (ELISA).

Results: HSV-1 reactivation induced stromal edema and opacification concomitantly with elevated activation of NF-kappaB and elevated production of TNF-alpha. Lornoxicam treatment significantly decreased the incidence of recurrent HSK, attenuated the corneal opacity scores, and also effectively suppressed both NF-kappaB activation and TNF-alpha expression in biological analysis. Histopathology examination revealed a reduced immunostaining positive cell density for NF-kappaB in the cornea from lornoxicam-treated mice as well as a diminished inflammatory response.

Conclusions: Lornoxicam exerts protective effects against HSK, presumably through the down-regulation of NF-kappaB activation.

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Related in: MedlinePlus

Electrophoretic mobility shift assessment of the DNA-binding activity of NF-κB in the cornea of different groups. A: Nuclear extracts were probed for NF-κB binding activity. Lane 1: mock-infected group (Control); lanes 2, 4, 6, 8, and10: Reactivated, saline-treated group (HSK) on days 4, 7, 10, 14, and 21, respectively; lanes 3, 5, 7, 9, and 11: Reactivated, LOR-treated group (HSK+LOR) on days 4, 7, 10, 14, and 21, respectively; lane 12: mock-infected group treated with lornoxicam (LOR). The data are representative of three independent experiments. B: Quantitative analysis is displayed of NF-κB activity by EMSA at days 4, 7, 10, 14, and 21 after irradiation in corneas of ICR mice treated with either saline or LOR. The bands were quantified using image analysis software (Bandleader 3.0 software, Magnitec Ltd. Tel Aviv, Israel). The relative intensity was determined by comparison with that of the background. The data are presented as mean±standard error of results from three independent experiments. EMSA shows a markedly upregulated activity of NF-κB in the HSK group and HSK+LOR group compared to levels in the control group, and this upregulation was dramatically suppressed by LOR treatment at each indicated time point. The asterisk indicates that p<0.05 when compared to the control group. The hash mark indicates that p<0.05 for the HSK+LOR group when compared to the HSK group at the corresponding time points.
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f2: Electrophoretic mobility shift assessment of the DNA-binding activity of NF-κB in the cornea of different groups. A: Nuclear extracts were probed for NF-κB binding activity. Lane 1: mock-infected group (Control); lanes 2, 4, 6, 8, and10: Reactivated, saline-treated group (HSK) on days 4, 7, 10, 14, and 21, respectively; lanes 3, 5, 7, 9, and 11: Reactivated, LOR-treated group (HSK+LOR) on days 4, 7, 10, 14, and 21, respectively; lane 12: mock-infected group treated with lornoxicam (LOR). The data are representative of three independent experiments. B: Quantitative analysis is displayed of NF-κB activity by EMSA at days 4, 7, 10, 14, and 21 after irradiation in corneas of ICR mice treated with either saline or LOR. The bands were quantified using image analysis software (Bandleader 3.0 software, Magnitec Ltd. Tel Aviv, Israel). The relative intensity was determined by comparison with that of the background. The data are presented as mean±standard error of results from three independent experiments. EMSA shows a markedly upregulated activity of NF-κB in the HSK group and HSK+LOR group compared to levels in the control group, and this upregulation was dramatically suppressed by LOR treatment at each indicated time point. The asterisk indicates that p<0.05 when compared to the control group. The hash mark indicates that p<0.05 for the HSK+LOR group when compared to the HSK group at the corresponding time points.

Mentions: To evaluate whether LOR diminished the activity of NF-κB, we performed EMSA to determine the levels of nuclear NF-κB DNA–binding activity in the cornea after recurrence. As shown in the EMSA blot, there was a constitutive low expression in the mock-infected group but prominent high intensity in the saline-treated, infected group (Figure 2). HSV-1 recurrence resulted in a significant upregulation of NF-κB activity, shown as a maximal fivefold induction of NF-κB compared with the mock-infected group. Moreover, an obvious decrease in NF-κB activity was found in the LOR-treated group compared to the saline-treated group. Since no differences were observed in NF-κB activity between the mock infected saline-treated group and the LOR alone group, it seemed that LOR alone did not have any significant effect on the activity of NF-κB.


Lornoxicam suppresses recurrent herpetic stromal keratitis through down-regulation of nuclear factor-kappaB: an experimental study in mice.

Yin J, Huang Z, Xia Y, Ma F, Zhang LJ, Ma HH, Li Wang L - Mol. Vis. (2009)

Electrophoretic mobility shift assessment of the DNA-binding activity of NF-κB in the cornea of different groups. A: Nuclear extracts were probed for NF-κB binding activity. Lane 1: mock-infected group (Control); lanes 2, 4, 6, 8, and10: Reactivated, saline-treated group (HSK) on days 4, 7, 10, 14, and 21, respectively; lanes 3, 5, 7, 9, and 11: Reactivated, LOR-treated group (HSK+LOR) on days 4, 7, 10, 14, and 21, respectively; lane 12: mock-infected group treated with lornoxicam (LOR). The data are representative of three independent experiments. B: Quantitative analysis is displayed of NF-κB activity by EMSA at days 4, 7, 10, 14, and 21 after irradiation in corneas of ICR mice treated with either saline or LOR. The bands were quantified using image analysis software (Bandleader 3.0 software, Magnitec Ltd. Tel Aviv, Israel). The relative intensity was determined by comparison with that of the background. The data are presented as mean±standard error of results from three independent experiments. EMSA shows a markedly upregulated activity of NF-κB in the HSK group and HSK+LOR group compared to levels in the control group, and this upregulation was dramatically suppressed by LOR treatment at each indicated time point. The asterisk indicates that p<0.05 when compared to the control group. The hash mark indicates that p<0.05 for the HSK+LOR group when compared to the HSK group at the corresponding time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697669&req=5

f2: Electrophoretic mobility shift assessment of the DNA-binding activity of NF-κB in the cornea of different groups. A: Nuclear extracts were probed for NF-κB binding activity. Lane 1: mock-infected group (Control); lanes 2, 4, 6, 8, and10: Reactivated, saline-treated group (HSK) on days 4, 7, 10, 14, and 21, respectively; lanes 3, 5, 7, 9, and 11: Reactivated, LOR-treated group (HSK+LOR) on days 4, 7, 10, 14, and 21, respectively; lane 12: mock-infected group treated with lornoxicam (LOR). The data are representative of three independent experiments. B: Quantitative analysis is displayed of NF-κB activity by EMSA at days 4, 7, 10, 14, and 21 after irradiation in corneas of ICR mice treated with either saline or LOR. The bands were quantified using image analysis software (Bandleader 3.0 software, Magnitec Ltd. Tel Aviv, Israel). The relative intensity was determined by comparison with that of the background. The data are presented as mean±standard error of results from three independent experiments. EMSA shows a markedly upregulated activity of NF-κB in the HSK group and HSK+LOR group compared to levels in the control group, and this upregulation was dramatically suppressed by LOR treatment at each indicated time point. The asterisk indicates that p<0.05 when compared to the control group. The hash mark indicates that p<0.05 for the HSK+LOR group when compared to the HSK group at the corresponding time points.
Mentions: To evaluate whether LOR diminished the activity of NF-κB, we performed EMSA to determine the levels of nuclear NF-κB DNA–binding activity in the cornea after recurrence. As shown in the EMSA blot, there was a constitutive low expression in the mock-infected group but prominent high intensity in the saline-treated, infected group (Figure 2). HSV-1 recurrence resulted in a significant upregulation of NF-κB activity, shown as a maximal fivefold induction of NF-κB compared with the mock-infected group. Moreover, an obvious decrease in NF-κB activity was found in the LOR-treated group compared to the saline-treated group. Since no differences were observed in NF-κB activity between the mock infected saline-treated group and the LOR alone group, it seemed that LOR alone did not have any significant effect on the activity of NF-κB.

Bottom Line: Lornoxicam treatment significantly decreased the incidence of recurrent HSK, attenuated the corneal opacity scores, and also effectively suppressed both NF-kappaB activation and TNF-alpha expression in biological analysis.Histopathology examination revealed a reduced immunostaining positive cell density for NF-kappaB in the cornea from lornoxicam-treated mice as well as a diminished inflammatory response.Lornoxicam exerts protective effects against HSK, presumably through the down-regulation of NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People's Republic of China.

ABSTRACT

Purpose: We designed the current study to determine the protective effects of lornoxicam, a cyclooxygenase (COX) inhibitor, on recurrent herpetic stromal keratitis (HSK) and the nuclear factor-kappaB (NF-kappaB)-mediated mechanism in mice.

Methods: A corneal latent herpes simplex virus-1 (HSV-1) infected mouse model was established. Six weeks later, Ultraviolet B (UVB) irradiation induced the recurrence. Corneal swabs were obtained and cultured with indicator cells to determine shedding of the virus. Lornoxicam was administered intraperitoneally daily, beginning one day before irradiation and lasting for seven days. Saline-treated and mock-infected control groups were also studied at the same time. Development of corneal inflammation and opacity was scored. Immunohistochemical staining and an electrophoretic mobility shift assay were performed to evaluate the effect of lornoxicam on NF-kappaB activation in the corneal tissues. The levels of tumor necrosis factor-alpha (TNF-alpha) in the cornea were determined by an enzyme-linked immunosorbent assay (ELISA).

Results: HSV-1 reactivation induced stromal edema and opacification concomitantly with elevated activation of NF-kappaB and elevated production of TNF-alpha. Lornoxicam treatment significantly decreased the incidence of recurrent HSK, attenuated the corneal opacity scores, and also effectively suppressed both NF-kappaB activation and TNF-alpha expression in biological analysis. Histopathology examination revealed a reduced immunostaining positive cell density for NF-kappaB in the cornea from lornoxicam-treated mice as well as a diminished inflammatory response.

Conclusions: Lornoxicam exerts protective effects against HSK, presumably through the down-regulation of NF-kappaB activation.

Show MeSH
Related in: MedlinePlus