Andrographolide attenuates bupivacaine-induced cytotoxicity in SH-SY5Y cells through preserving Akt/mTOR activity

Abstract

Background: Bupivacaine (Bup) is the most commonly used local anesthetic. However, Bup induces cytotoxicity, especially in older patients. Recent reports have indicated that andrographolide (Andro) exhibits protective effects on human neurons. Nevertheless, whether Andro can inhibit Bup-induced cytotoxicity remains unclear. As such, we investigated the effect of Andro on Bup-induced cytotoxicity of SH-SY5Y cells in the present study. Methods: Western blotting was used to examine expression of Bax, Bcl2, active caspase 3, p-Akt, and p-mTOR in SH-SY5Y cells. In addition, ELISA was used to detect levels of total glutathione and reactive oxygen species in cells. Results: We found that Andro attenuated Bup-induced cytotoxicity of SH-SY5Y cells. In addition, Andro inhibited Bup-induced apoptosis via downregulating the expression of Bax and active caspase 3 and upregulating the proteins Bcl2, p-Akt, and p-mTOR in SH-SY5Y cells. Moreover, Andro alleviated Bup-induced oxidative damage in SH-SY5Y cells via downregulating the level of reactive oxygen species and upregulating of the level of total glutathione. More significantly, inhibition of Akt abolished the protective effect of Andro in Bup-treated SH-SY5Y cells. Conclusion: Our findings indicated that Andro played a neuroprotective role via preserving Akt/mTOR activity and increasing antioxidative status in Bup-treated SH-SY5Y cells. Therefore, Andro may be a potential agent for the treatment of human cytotoxicity induced by Bup.

Keywords: Akt; andrographolide; apoptosis; bupivacaine; cytotoxicity.

Figure 1

Inhibitory effects of Bup or Andro on SH-SY5Y cell growth.Notes: (A) Chemical structure of Andro. (B) SH-SY5Y cells were exposed to Bup (0, 200, 400, 600, or 1,000 μM) for 48 hours. Then, cell viability was determined using CCK8 assay. (C) SH-SY5Y cells were exposed to Andro (0, 50, 100, 200, or 400 μM) for 12 hours. Then, cell viability was determined using CCK8 assay. *P<0.05; **P<0.01.Abbreviations: Bup, bupivacaine; Andro, andrographolide..

Figure 2

Andro alleviated Bup-induced cytotoxicity via inhibition of apoptosis in SH-SY5Y cells.Notes: (A) SH-SY5Y cells were incubated with Andro (0, 50, 100 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. Cell viability was detected with CCK8 assay. (B) SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. Proliferation of SH-SY5Y cells was determined using Ki67 staining. Representative fluorescence images of Ki67 and DAPI staining (mgnification 200×). (C) Ki67 positive cells in each group was counted. (D) Cell apoptosis in SH-SY5Y cells were detected by annexin V–PI staining. (E) The cell-apoptosis rate in each group was calculated. **P<0.01.Abbreviations: Bup, bupivacaine; Andro, andrographolide.

Figure 3

Andro attenuated Bup-induced cytotoxicity via increasing anti-oxidative status in SH-SY5Y cells.Notes: SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with BUP (0 or 400 μM) for another 48 hours. Production of ROS and total GSH SH-SY5Y cells were detected with ELISA kits. **P<0.01.Abbreviations: Bup, bupivacaine; Andro, andrographolide; ROS, reactive oxygen species; GSH, glutathione.

Figure 4

Andro attenuated Bup-induced cytotoxicity via activating p-Akt and p-mTOR in SH-SY5Y cells.Notes: SH-SY5Y cells were incubated with Andro (0, 50, 100, or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. (A) Expression of Bax, Bcl2, active caspase 3, p-Akt, and p-mTOR in Bup-induced SH-SY5Y cells was analyzed by Western blotting. (B) Relative Bax expression was quantified by normalizing to β-actin. (C) Relative Bcl2 expression was quantified by normalizing to β-actin. (D) Relative active caspase 3 expression was quantified by normalizing to β-actin. (E) Relative p-Akt expression was quantified by normalizing to β-actin. (F) Relative p-mTOR expression was quantified by normalizing to β-actin. **P<0.01.Abbreviations: Bup, bupivacaine; Andro, andrographolide.

Figure 5

An Akt inhibitor abrogated the protective effect of Andro in Bup-treated SH-SY5Y cells. Notes: SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with 10 nM AZD5363 for 1 hour. Later, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. (A) Proliferation of SH-SY5Y cells was determined using CCK8 assay. (B) Apoptotic cells were detected with annexin V–PI staining. (C) Cell-apoptosis rates were calculated. **P<0.01. Abbreviations: Bup, bupivacaine; Andro, andrographolide.

Figure 6

An Akt inhibitor abrogated the protective effect of Andro in Bup-treated SH-SY5Y cells via inhibition of the Akt–mTOR pathway.Notes: SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with 10 nM AZD5363 for 1 hour. Later, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. (A) Expression of p-Akt, p-mTOR, and active caspase 3 in SH-SY5Y cells was analyzed by Western blotting. (B) Relative p-Akt expression was quantified by normalizing to β-actin. (C) Relative p-mTOR expression was quantified by normalizing to β-actin. (D) Relative active caspase 3 expression was quantified by normalizing to β-actin. (E) Representative fluorescence images of p-AKt and DAPI (magnification 400×). **P<0.01.Abbreviations: Bup, bupivacaine; Andro, andrographolide.