Effect of Tetramethylpyrazine on RPE degeneration, choroidal blood flow and oxidative stress of RPE cells

Abstract

Aim: To study the effects of Tetramethylpyrazine (TMP) on retinal pigment epithelium (RPE) degeneration, choroidal blood flow and oxidative stress of RPE cells.

Methods: The 35mg/kg NaIO(3)-induced RPE degeneration rat eyes was given 25µg 1% TMP eye drops 3 times a day for 7 days before NaIO(3) injection, and then 2 to 4 weeks after NaIO(3) injection. RPE function was measured with c-wave of electroretinogram (ERG). Colored microsphere technique was used for in vivo experiments to determine the choroidal blood flow in ocular hypertensive (40mmHg) rabbit eyes. Methylthiazoltetrazolium (MTT) assay was used to study in vitro effect of TMP on various oxidants induced injury in the hRPE (ARPE-19 (ATCC, Manassas, VA, USA)).

Results: Two weeks after NaIO(3) injection, the amplitude of ERG c-wave fell markedly in NaIO(3) group to 36% of control group(P<0.01). No apparent difference was observed in TMP+NaIO(3) group. Four weeks later, the NaIO(3) group fell to 46% of control group (P<0.01), while the TMP+NaIO(3) group fell to only 77% of control group (P<0.01). There was a 67% reversal of the ERG c-wave by TMP as compared to NaIO(3) group(P<0.01). The choroidal blood flow was significantly increased at all time points (at 30, 60 and 120 minutes after TMP instillation) as compared with corresponding controls. TMP had no effect on hypoxia-(1% O(2)), t-BHP- and H(2)O(2)-induced damage in RPE cells. 10(g/mL TMP could reverse 1 and 3mM NaN(3)-induced loss of viability of RPE by 18.5% (P<0.01) and 23% (P<0.01), respectively. 30µg/mL TMP could reverse 30 and 100mM NaIO(3) induced loss of viability of RPE by 18.1% (P<0.05) and 16.8% (P<0.01), respectively.

Conclusion: TMP can significantly protect RPE from NaIO(3) induced degeneration in vivo and oxidative stress in vitro and can increase choroidal blood flow markedly in vivo.

Keywords: Tetramethylpyrazine; age-related macular degeneration; choroidal blood flow; oxidative stress; retinal pigment epithelium; sodium iodate.

Figure 1. A: effect of TMP on NaIO₃ induced RPE degeneration in rat eyes for 2 weeks. a: control group compares with NaIO₃ group (P=4.83E-9). b: NaIO₃+0.5%TMP group compares with NaIO₃ group(P=9.78E-5); B: effect of TMP on NaIO₃ induced RPE degeneration in rat eyes for 4 weeks. a: control group compares with NaIO₃ group(P=8.98E-6). b: NaIO₃+0.5%TMP group compares with NaIO₃ group(P=3.92E-5)

Figure 2. Effect of TMP on the choroid blood flow of rabbit eyes

^aP<0.05 and ^bP<0.01 vs vehicle control group. The data was expressed as mean±SEM, in the vehicle control group n =7 and in the TMP group n =6

Figure 3. Effect of TMP on the proliferation of RPE cells

RPE cells were incubated with TMP for 48 hours

Figure 4. Effect of TMP on NaN₃-induced ischemia toxicity in RPE cells

A: effect of NaN₃ on the proliferation of RPE cells; B: Effect of TMP on NaN₃-induced ischemia toxicity in RPE cells. RPE cells were incubated with TMP and NaN₃ for 48 hours. ^aP<0.05 and ^bP<0.01 vs vehicle control. ^dP<0.01 vs model control. The data was expressed as mean±EM, n=6

Figure 5. Effect of TMP on NaIO₃-induced injury in RPE cells

A: effect of NaIO₃ on the proliferation of RPE cells; B: effect of TMP on NaIO₃-induced injury in RPE cells. RPE cells were incubated with TMP and NaIO₃ for 48 hours. ^aP<0.05 and ^bP<0.01 vs vehicle control. ^cP<0.05 and ^dP<0.01 vs model control. The data was expressed as mean±SEM, n =6