Hydrogen Sulfide Inhibits Morphine-induced Oxidative Stress And Its Mechanism

Drug dependence is a universal social problem that plagues all ethnic groups and classes around the world.The data show that a considerable number of patients will die due to an overdose of drugs,causing serious problems to families and society.Morphine is a common abused drug.It has been reported that long-term use of morphine induces oxidative stress,leading to neuronal apoptosis and brain damage.Studies have shown that hydrogen sulfide,as a novel gas signaling molecule and neuromodulation,can play an anti-apoptotic and anti-oxidative role,thereby reducing brain damage caused by drug dependence.At present,there are still many unresolved questions about the morphine addiction mechanism.Whether hydrogen sulfide can reduce or inhibit morphine dependence by inhibiting oxidative stress is still unclear.In this study,we established a mouse morphine dependence model to investigate the effect of hydrogen sulfide on the morphine-induced addictive behaviors,and further used SH-SY5Y cells to establish a morphine-induced oxidative stress model to explore the mechanism underpinning antioxidant stress of hydrogen sulfide.The main research contents and results are as follows:(1)Male C57/BL6 mice(20-25 g)aged 8-10 weeks were used to establish a morphine-dependent animal model.The mice were randomly divided into three groups,control group,morphine group,and hydrogen sulfide+morphine group.Mice of the morphine group were injected with hydrochloric acid morphine subcutaneously twice a day(12-hours interval)for 4 consecutive days.The daily doses of morphine were set at 20 mg/kg initially and increased with a step of 10 mg/kg in the following days.Mice of hydrogen sulfide+morphine group were treated with sodium hydrosulfide(5.6 mg/kg)intraperitoneally 30 minutes before each injection of morphine.On day 5,animals received a single morphine(20 mg/kg)injection at 8:00 am and a single intraperitoneal injection of naloxone(10 mg/kg)after 4 hours to induce withdrawal behaviors.Immediately thereafter,each mouse was placed in an acrylic glass box(30 × 30 × 40 cm),and the number of jumps within 15 minutes was recorded as an observational indicator of morphine withdrawal symptoms.The body weight of the mice was measured before and 30 minutes after the injection of naloxone.The results showed that hydrogen sulfide pretreatment significantly reduced the number of jumps induced by naloxone,indicating that hydrogen sulfide can relieve withdrawal symptoms after morphine dependence.(2)SH-SY5Y cells were selected to establish a morphine-induced oxidative stress model,which was divided into three groups:control group,morphine group and hydrogen sulfide+morphine group.Intracellular ROS levels were measured by using 2’,7’-dichlorodihydrofluorescein diacetate as a fluorescent probe.The results showed that treatment with morphine(25 μM and 50 μM)for 24 h caused an increase in intracellular ROS concentration and vulcanization.Hydrogen sulfide(100 μM)pretreatment for 30 min can significantly reduce ROS production.At the same time,the intracellular MDA production and SOD activity were measured.It was found that morphine could increase the MDA content and decrease the SOD activity in the cells,compared with the control group,indicating that morphine destroys the balance of the antioxidant enzyme system and weakens the cell protection.Hydrogen sulfide pretreatment can significantly reduce intracellular MDA production and enhance SOD activity,indicating that hydrogen sulfide has a significant protective effect on oxidative stress induced by morphine.(3)To further detect the antioxidative effect of hydrogen sulfide,the expression level of Cytochrome-C in cells was detected by Western blotting.The results showed that the expression of Cytochrome-C in cells increased in a time-dependent manner.Hydrogen sulfide pretreatment can significantly reduce the expression of Cytochrome-C.Subsequently,JC-1 and Hoechst 33258 were used as fluorescent probes to detect changes in mitochondrial membrane potential and degree of apoptosis,respectively.The results showed that morphine exposure resulted in the loss of mitochondrial membrane potential and apoptosis.Hydrogen sulfide pretreatment significantly increased mitochondrial membrane potential and inhibited apoptosis.LDH test results showed that morphine exposure leads to increased LDH release and enhances cytotoxicity,while hydrogen sulfide pretreatment can significantly inhibit LDH release,reduce cytotoxicity,and play a cytoprotective role.(4)To explore the mechanism of the protective effect of hydrogen sulfide on morphine-induced oxidative stress,the expression levels of P66Shc and Nrf2/Keap1 pathway proteins were detected by Western blotting.It was found that morphine can activate the Nrf2/Keapl signaling pathway in the cell,promote the uncoupling of Nrf2 protein and Keapl protein into the nucleus,and hydrogen sulfide can significantly inhibit these effects,indicating that the Nrf2/Keapl signaling pathway is involved in morphine caused cellular oxidative stress and hydrogen sulfide can inhibit the activation of the Nrf2/Keap1 signaling pathway.In summary,this study suggests that hydrogen sulfide can inhibit morphine-induced oxidative stress and inhibit apoptosis by inhibiting the activation of the Nrf2/Keap1 signaling pathway.This may provide new ideas for the treatment of morphine dependence.