Background
Neurotoxicity induced by local anesthetics (LAs) is potentially life threatening, especially for patients with underlying diseases like diabetes. The anesthetic bupivacaine (Bup) has been ...reported to induce neurotoxicity mediated by reactive oxygen species (ROS), which is aggravated by hyperglycemia. Krüppel-like factor 9 (KLF9), an axon growth-suppressing transcription factor, plays a key role in neuronal maturation and promotes oxidative stress. This study was designed to investigate whether and how KLF9 regulates ROS levels related to LA neurotoxicity under hyperglycemic conditions.
Methods
Klf9/GFP ShRNA (LV Sh-
Klf9
) was used to achieve stable
Klf9
knockdown in the SH-SY5Y cell line. KLF9-deficient and normal cells were cultured under normal or high-glucose (HG) culture conditions and then exposed to Bup. Cell viability, intracellular and mitochondrial ROS, and mitochondrial membrane potential (ΔΨm) were detected to examine the role of KLF9. Thereafter, KLF9-deficient and normal cells were pretreated with small-interfering RNA targeting peroxiredoxin 6 (siRNA-
Prdx6
) to determine if PRDX6 was the target protein in HG-aggravated Bup neurotoxicity.
Results
The mRNA and protein levels of KLF9 were increased after Bup and hyperglycemia treatment. In addition, cell survival and mitochondrial function were significantly improved, and ROS production was decreased after Sh-
Klf9
treatment compared with Sh-Ctrl. Furthermore, the expression of PRDX6 was suppressed by Bup in hyperglycemic cultures and was upregulated in the
Sh-Klf9
group. Moreover, the protection provided by KLF9 deficiency for cell survival, the increase in ROS production in cells and mitochondria, and the disruption of mitochondrial function were abolished by
Prdx6
knockdown.
Conclusions
The results of this study demonstrated that hyperglycemia aggravated Bup neurotoxicity by upregulating KLF9 expression, which repressed the antioxidant PRDX6 and led to mitochondrial dysfunction, ROS burst, and cell death. Understanding this mechanism may, thus, offer valuable insights for the prevention and treatment of neurotoxicity induced by LAs, especially in diabetic patients.
Opioid‐induced hyperalgesia (OIH) and allodynia is a well‐known phenomenon and refers to the pain sensitization in patients after prolonged opioid exposure. OIH limits the use of opioids in pain ...control, but the underlying mechanisms are not fully clear. This study investigated the role of mitochondrial Ca2+ uniporter (MCU) in remifentanil (a commonly used opioid analgesic)‐induced allodynia. Using a rat model of OIH, we found that incision‐ and remifentanil‐induced mechanical allodynia were remarkably attenuated by pretreatment with Ru360, a specific MCU antagonist, suggesting a critical role of MCU in both incision‐ and opioid‐induced allodynia. In addition, imaging studies with Rhod‐2 (a mitochondrial Ca2+ dye) in spinal tissues demonstrated increased mitochondrial Ca2+ level in response to incision and remifentanil infusion, which was attenuated by Ru360. Western blot and immunohistochemistry showed that pNR phosphorylated N‐methyl‐D‐aspartate (NMDA) receptor and pERK (phosphorylated extracellular signal‐regulated kinase) are increased during both incision‐induced hyperalgesia and remifentanil‐induced hyperalgesia, and again the increases in pNR and pERK were remarkably attenuated by Ru360. Together, our data demonstrate that MCU plays a critical role in remifentanil‐induced postoperative mechanical allodynia, with NMDA receptor and ERK as possible downstream effectors. Our findings provide novel mechanisms for remifentanil‐induced mechanical allodynia and encourage future studies to examine the mitochondrial Ca2+ uniporter as a potential therapeutic target for prevention of OIH.
This study demonstrates that mitochondrial Ca2+ uniporter (MCU) plays a critical role in remifentanil‐induced postoperative mechanical allodynia. Our results indicate that NMDA receptor and ERK are possible effectors downstream to MCU.
Local anesthesia has been recommended for percutaneous endoscopic lumbar discectomy (PELD) in recent years; however, the efficacy, including oxidative stress, inflammatory reactions and ventilation ...effects, when intravenous dexmedetomidine (DEX) is administered during PELD has not been described.
Sixty adult patients undergoing PELD were randomly allocated to either an intravenous DEX sedation group (Group A) or a normal saline group (Group B). Respiratory data, including minute ventilation (MV), tidal volume (TV), and respiratory rate (RR), were recorded using a respiratory volume monitor (RVM), and peripheral oxygen saturation (SpO
) was monitored by pulse oximetry. The visual analog score (VAS) was used to assess the level of pain. The serum levels of inflammatory biomarkers including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were to assess inflammatory reactions. The serum levels of oxidative stress biomarkers including malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) were also recorded to evaluate oxidative stress.
There were no significant differences in RR, MV, TV and SpO
between the two groups at any time point (P > 0.05). Group B exhibited lower serum levels of GSH-PX (P < 0.0001) and higher serum levels of MDA (p < 0.0001) than Group A at the end of surgery. Twenty-four hours after surgery, Group B exhibited higher serum levels of IL-6 (P = 0.0033), TNF-α (P = 0.0002), and MDA (P < 0.0001) and lower serum levels of GSH-PX (P < 0.0001) than Group A. In addition, Group A exhibited lower VAS (P < 0.0001) than Group B during surgery.
DEX administration using RVM not only provides analgesia without ventilatory depression but also alleviates oxidative stress and inflammatory reactions in patients undergoing PELD.
Bupivacaine has been widely used in clinical Anesthesia, but its neurotoxicity has been frequently reported, implicating cellular oxidative DNA damage as the major underlying mechanism. However, the ...mechanism underlying bupivacaine-induced oxidative DNA damage is unknown. We, thus, exposed SH-SY5Y cells to 1.5mM bupivacaine to induce neurotoxicity. Then, iTRAQ proteomic analysis was used to explore the repair of neuronal oxidative DNA damage. By analyzing the STRING version 11.0 database, the bioinformatics relationship between key repair enzymes was tracked. Subsequently, immunofluorescence co-localization and immunoprecipitation were used to investigate the interaction between key repair enzymes. The iTRAQ showed that Poly ADP-ribose polymerase 1 (PARP-1) from the base excision repair pathway participated closely in the repair of oxidative DNA damage induced by bupivacaine, and inhibition of PARP-1 expression significantly aggravated bupivacaine-induced DNA damage and apoptosis. Interestingly, this study showed that there were interactions and co-expression between PARP-1 and XPD (xeroderma pigmentosum D), another key protein of the nucleic acid excision repair pathway. After inhibiting XPD, PARP-1 expression was significantly reduced. However, simultaneous inhibition of both XPD and PARP-1 did not further increase DNA damage. It is concluded that PARP-1 may repair bupivacaine-induced oxidative DNA damage through XPD-mediated interactions.
Bupivacaine (BP) is a commonly clinically used local anesthetic (LA). Current studies suggest that neurological complications are increased in diabetic patients after LA application, but the ...molecular mechanism is poorly understood. LA-induced autophagy and neuronal injury have been reported. We hypothesized that a high-glucose environment aggravates BP-induced autophagic damage. Mouse dorsal root ganglion (DRG) neurons were treated with BP in a high-glucose environment, and the results showed that reactive oxygen species (ROS) levels increased, autophagy was activated, autophagy flux was blocked, and cell viability decreased. Pretreatment with the ROS scavenger N-acetyl-cysteine (NAC) attenuated ROS-mediated autophagy regulation. Moreover, the expression of the long noncoding RNA (lncRNA) taurine upregulated gene 1 (TUG1) increased, and NAC and TUG1 siRNA inhibited the expression of TUG1/mammalian target of rapamycin (mTOR) in DRGs treated with BP in a high-glucose environment. Intriguingly, contrary to previous reports on a positive effect on neurons, we found that rapamycin, an autophagy activator, and chloroquine, an autophagy and lysosome inhibitor, both exacerbated autophagic damage. These data suggest that a high-glucose environment exacerbated BP induced ROS-dependent autophagic damage in DRG neurons through the TUG1/mTOR signaling pathway, which provides a theoretical basis and target for the clinical prevention and treatment of BP neurotoxicity in diabeties.
The research aimed to explore the biological role of p53 protein and long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in bupivacaine (bup)-induced neurotoxicity. Our work treated dorsal ...root ganglion (DRG) cells with bup, detected cell viability through CCK-8, apoptosis through TUNEL assays, DeoxyriboNucleic Acid (DNA) damage through γ-H2AX protein and comet assay, including p53 mRNA, protein and TUG1 expression through q-PCR and western blot, furthermore, cell viability and DNA damage were determined after the silencing of p53 and TUG1, biological information and TUG1 FISH combined with p53 protein immunofluorescence (IF) was performed to determine the cellular localization of these molecule. In vivo experiments, we explored the impact of intrathecal injection of bup on p53 mRNA and protein, TUG1, γ-H2AX protein expression. The results showed that bup was available to signally decreased cell viability, promoted apoptosis rate and DNA damage, additionally, bup increased p53 mRNA and protein and TUG1 expression. P53 siRNA and TUG1 siRNA significantly increased DNA damage. Furthermore, bioinformatics analysis and colocalization experiments revealed that the p53 protein is a transcription factor of TUG1, in vivo experiment, intrathecal injection of bup increased the p53 mRNA, p53 protein, TUG1 and γ-H2AX protein in the murine DRG. In this study, it was found p53 and TUG1 promote the repair of the DNA damage induced by bup in murine dorsal root ganglion cells, suggesting a new strategy for the amelioration of bup-induced neurotoxicity.
Bupivacaine, a local anesthetic widely used for regional anesthesia and pain management, has been reported to induce neuronal injury, especially DNA damage. Neurons employ different pathways to ...repair DNA damage. However, the mechanism underlying bupivacaine-mediated DNA damage repair is unclear. A rat neuronal injury model was established by intrathecal injection of (3%) bupivacaine. An in vitro neuronal injury model was generated by exposing SH-SY5Y cells to bupivacaine (1.5 mmol/L). Then, a cDNA plate array was used to identify the DNA repair genes after bupivacaine exposure. The results showed that xeroderma pigmentosum complementary group D (XPD) of the nuclear excision repair (NER) pathway was closely associated with the repair of DNA damage induced by bupivacaine. Subsequently, Western blot assay and immunohistochemistry indicated that the expression of the repair enzyme XPD was upregulated after DNA damage. Downregulation of XPD expression by a lentivirus aggravated the DNA damage induced by bupivacaine. In addition, phosphatidyl-3-kinase (PI3K)/AKT signaling in neurons was inhibited after exposure to bupivacaine. After PI3K/AKT signaling was inhibited, bupivacaine-mediated DNA damage was further aggravated, and the expression of XPD was further upregulated. However, knockdown of XPD aggravated bupivacaine-mediated neuronal injury but did not affect PI3K/AKT signaling. In conclusion, the repair enzyme XPD, which was partially regulated by PI3K/AKT signaling, responded to bupivacaine-mediated neuronal DNA damage. These results can be used as a reference for the treatment of bupivacaine-induced neurotoxicity.
Opioid-induced hyperalgesia and allodynia (OIH) is a well-known phenomenon and refers to the pain sensitization in patients after prolonged opioid exposure. OIH limits the use of opioids in pain ...control, but the underlying mechanisms are not fully clear. The present study investigated the role of mitochondrial Ca
uniporter (MCU) in remifentanil (a commonly used opioid analgesic)-induced allodynia. Using a rat model of OIH, we found that incision- and remifentanil-induced mechanical allodynia were remarkably attenuated by pretreatment with Ru360, a specific MCU antagonist, suggesting a critical role of MCU in both incision- and opioid-induced allodynia. In addition, imaging studies with Rhod-2 (a mitochondrial Ca
dye) in spinal tissues demonstrated increased mitochondrial Ca
level in response to incision and remifentanil infusion, which was attenuated by Ru360. Western blot and immunohistochemistry showed that pNR (phosphorylated N-methyl-D-aspartate (NMDA) receptor) and pERK (phosphorylated extracellular signal-regulated kinase) are increased during both incision- and remifentanil-induced hyperalgesia, and again the increases in pNR and pERK were remarkably attenuated by Ru360. Together, our data demonstrate that MCU plays a critical role in remifentanil-induced postoperative mechanical allodynia, with NMDA receptor and ERK as possible downstream effectors. Our findings provide novel mechanisms for remifentanil-induced mechanical allodynia, and encourage future studies to examine the mitochondrial Ca
uniporter as a potential therapeutic target for prevention of OIH. This article is protected by copyright. All rights reserved.
•High glucose causes an increase in cell toxicity induced by BP.•High glucose aggravated BP-induced DNA damage and apoptosis.•High glucose aggravated BP-induced DNA damage through inhibit DNA-PK/Ku70 ...protein.
Bupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression.
We examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting.
Compared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions.
These data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.
PDF
