Cascade Signals of Papaverine Inhibiting LPS-Induced Retinal Microglial Activation

Abstract

Studies have shown that papaverine can inhibit lipopolysaccharide (LPS)-induced microglial activation. The retinal primary microglia of newborn SD rats were isolated and purified, and a LPS-induced microglia activation model was established. The protein phosphorylation level of the signaling pathway was detected by western blotting. The transcription and expression of TNF-α, IL-1β, and IL-10 were respectively detected by RT-PCR and ELISA to observe the abnormal activation of primary microglia. The cAMP inhibitor Rp-isomer, PKA inhibitor H89, and MEK inhibitor U0126 were separately added to further investigate the role of MEK/Erk in PAP inhibition of primary microglial activation and the relationship between cAMP/PKA and MEK/Erk. It was found that the level of MEK phosphorylation was upregulated after LPS stimulation, which was blocked by 10 μg/ml of papaverine.10μM U0126 significantly inhibited TNF-α and IL-1β and increased IL-10 transcription and expression in retinal microglia (P < 0.01). Both Rp-isomer and H89 upregulated the phosphorylation levels of MEK and Erk. Papaverine may inhibit inflammatory factors and promote the expression of anti-inflammatory factors through the cAMP/PKA and MEK/Erk pathway, thereby inhibiting LPS-induced activation of primary retinal microglia, and the MEK/Erk pathway may be partially regulated by cAMP/PKA, which can provide theoretical basis and experimental basis for its protection of the central nervous system.

Keywords: MEK/Erk; Papaverine; Primary microglia; cAMP/PKA.

Fig. 1

The transcription and expression of TNF-α and IL-1β after papaverine pretreatment. Primary microglia were pretreated with papaverine (0, 0.4, 2, and 10 μg/ml) for 4 h and incubated with LPS (100 ng/ml) for another 24 h. a The transcription of TNF-α and IL-1β were detected by RT-PCR (n = 5). b The expression of TNF-α and IL-1β were detected by ELISA (n = 5). *P < 0.05 versus LPS group, **P < 0.01 versus LPS group

Fig. 2

The transcription and expression of IL-10 after papaverine pretreatment. Primary microglia were pretreated with papaverine (0, 0.4, 2, and 10 μg/ml) for 4 h and stimulated by LPS for another 1 day. The transcription and expression of IL-10 were detected by RT-PCR and ELISA, respectively (n = 5). *P < 0.05 versus LPS group, **P < 0.01 versus LPS group

Fig. 3

Papaverine suppresses TNF-α and IL-1β by cAMP/PKA signaling pathway. a Treatment with 10 μg/ml of papaverine significantly upregulates cAMP (n = 5, P < 0.01 compared with CON group), while LPS can partly inhibit the increase of cAMP (n = 5, P < 0.01 compared with PAP group; P < 0.05 compared with CON group). ##P < 0.01 versus CON group, **P < 0.01 versus PAP group. b Primary retinal microglia were pretreated with 200 μmol Rp-isomer and 5 μmol H89 for 30 min, then treated with 10 μg/ml of papaverine for 4 h, and finally incubated with 100 ng/ml LPS for 1 h. The expression level of TNF-α and IL-lβ were detected by ELISA. The results showed that papaverine could inhibit the expression of TNF-α and IL-1β which upregulated by LPS (n = 5). After adding Rp-isomer, the expression of TNF-α and IL-1β were increased (n = 5). Similarly, the expression of TNF-α and IL-1β were increased after adding H89 (n = 5). ##P < 0.01 versus CON group, **P < 0.01 versus LPS group, ++ P < 0.01 versus LPS + PAP group

Fig. 4

a–c Papaverine inhibited phosphorylation of MEK and Erk in LPS-induced microglia. Primary retinal microglia were pretreated with 10 μg/ml papaverine for 4 h, and 100 ng/ml of LPS was added for further 1 h. The cells were lysed, total protein was extracted, and the expression of phosphorylated MEK and Erk were detected by Western Blotting. The results showed that LPS significantly upregulated the phosphorylation levels of MEK and Erk, and papaverine blocked the above effects (n = 4). ##P < 0.01 versus CON group, **P < 0.01 versus LPS group

Fig. 5

a, b U0126 inhibited the transcription and expression of TNF-α and IL-1β. Primary retinal microglia were pretreated with 10 μmol U0126 for 1 h, then treated with 10 μg/ml of papaverine for 4 h, and finally added with 100 ng/ml LPS for 1 h. The cells were harvested, RNA was extracted, the cDNA was synthesized, and the concentration of TNF-α mRNA and IL-1β mRNA were detected by RT-PCR. The supernatant was collected and the concentrations of TNF-α and IL-1β were detected by ELISA. The results showed that U0126 significantly inhibited the transcription (a) and expression (b) of TNF-α and IL-1β that upregulated by LPS (n = 5, P < 0.01 compared with the LPS group). **P < 0.01 versus LPS group

Fig. 6

U0126 increased the transcription and expression of IL-10. Primary retinal microglia were pretreated with 10 μmol of U0126 for 1 h, then treated with 10 μg/ml of papaverine for 4 h, and finally added with 100 ng/ml of LPS for 1 h. Cells were harvested and RNA was extracted. The cDNA was synthesized and the concentration of IL-10 mRNA was detected by RT-PCR. The supernatant was collected and the concentration of IL-10 was detected by ELISA. The results showed that U0126 can further upregulate the transcription and expression of IL-10(n = 5, P < 0.01 compared with the LPS group). *P < 0.05 versus LPS group, **P < 0.01 versus LPS group

Fig. 7

a–c H89 and Rp-isomer blocked the inhibition of papaverine on the phosphorylated MEK and Erk in LPS-induced microglia. Primary retinal microglia were pretreated with 200 μmol Rp-isomer and 5 μmol H89 for 30 min, then treated with 10 μg/ml of papaverine for 4 h, and finally incubated with 100 ng/ml LPS for 1 h. The cells were lysed, total protein was extracted, and the expression of phosphorylated MEK and Erk were detected by Western Blotting. The results showed that papaverine could block the phosphorylation of MEK and Erk that upregulated by LPS, while Rp-isomer and H89 partly blocked these effects caused by papaverine (n = 6). ##P < 0.01 versus CON group, **P < 0.01 versus LPS group, ++ P < 0.01 versus LPS+PAP group