The impact of oxidative stress-induced mitochondrial dysfunction on diabetic microvascular complications

Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia, with absolute insulin deficiency or insulin resistance as the main cause, and causes damage to various target organs including the heart, kidney and neurovascular. In terms of the pathological and physiological mechanisms of DM, oxidative stress is one of the main mechanisms leading to DM and is an important link between DM and its complications. Oxidative stress is a pathological phenomenon resulting from an imbalance between the production of free radicals and the scavenging of antioxidant systems. The main site of reactive oxygen species (ROS) production is the mitochondria, which are also the main organelles damaged. In a chronic high glucose environment, impaired electron transport chain within the mitochondria leads to the production of ROS, prompts increased proton leakage and altered mitochondrial membrane potential (MMP), which in turn releases cytochrome c (cyt-c), leading to apoptosis. This subsequently leads to a vicious cycle of impaired clearance by the body’s antioxidant system, impaired transcription and protein synthesis of mitochondrial DNA (mtDNA), which is responsible for encoding mitochondrial proteins, and impaired DNA repair systems, contributing to mitochondrial dysfunction. This paper reviews the dysfunction of mitochondria in the environment of high glucose induced oxidative stress in the DM model, and looks forward to providing a new treatment plan for oxidative stress based on mitochondrial dysfunction.

The concept of oxidative stress was first introduced by the German scientist Helmut Sies and refered to the imbalance of oxidants and antioxidants that can cause damage to the organism ( 19 ). The concept of “oxidative stress” was later extended to refer to a pathological phenomenon resulting from an imbalance between the production of free radicals and the scavenging function of the antioxidant system, and is closely linked to the development of diseases such as chronic obstructive pulmonary disease, Alzheimer’s disease, cancer, DM, hypertension and age-related diseases ( 20 – 26 ). Structurally, free radicals are highly reactive substances containing at least one unpaired electron and are active derivatives of ROS and reactive nitrogen species (RNS), which are closely associated with the initiation of oxidative stress ( 27 , 28 ). Both endogenous and exogenous stimulation lead to pathological increases in ROS and promote oxidative stress. Endogenous factors such as metabolic factors, mitochondrial damage, immune system dysregulation, and inflammatory products induce the production of ROS ( 29 – 32 ). Exogenous ionizing radiation, xenobiotics, ultraviolet light, alcohol abuse and smoking contribute to the onset and progression of aging and metabolic disease by promoting ROS production in the body ( 33 – 38 ) ( Figure 1 ). Figure 1 Endogenous and exogenous factors in ROS production. ROS generation is divided into two aspects: endogenous pathogenic factors and exogenous pathogenic factors. Endogenous pathogenic factors include mitochondrial disorder, metabolism, inflammatory products, immune system, etc. Exogenous pathogenic factors include radiation, xenobiotics, smoking, alcohol abuse, etc. ROS, reactive oxygen species. The intracellular ROS is mainly composed of O 2 .- [reduced from O 2 by electrons through the electron transfer chain (ETC)] and its derivatives. The three main reactive substances of ROS are superoxide anion (O 2 .- ), hydroxyl radical (•OH) and hydrogen peroxide (H 2 O 2 ), with •OH being the most reactive ( 39 , 40 ). Superoxide dismutase (SOD), the first line of defense of the cellular antioxidant defense system, promotes the production of the superoxide O 2 .- intermediating H 2 O 2 : 2O 2 .- +2H + →H 2 O 2 +O 2 ( 41 ). Subsequently reduced by catalase (CAT) to non-toxic H 2 O: 2H 2 O 2 →2H 2 O+O 2 ; or catalyzed by glutathione peroxidase (GSH-Px) to produce H 2 O: 2GSH+H 2 O 2 →2H 2 O+GSSG, all of which complete the antioxidant scavenging process and constitute the antioxidant enzymatic response system of the organism, maintaining the intracellular redox balance ( 41 , 42 ). Fe 2+ and Cu + are capable of redox reactions with unpaired electrons, destroying the structure and function of proteins, nucleic acids and lipids and cross-linking with these macromolecules to produce toxic substances ( 19 ). Activation of mitochondrial permeability transition pore (MPTP) promotes the release of ROS ( 43 ). Also, the vicious cycle of oxidative stress is facilitated by an increase in toxic substances such as malondialdehyde (MDA), and 4-hydroxy-2-nonenal (4-HNE), which are lipid peroxidation products ( 44 , 45 ). Likewise, advanced glycosylation end products (AGEs) further promote ROS production, induce overexpression of endothelial angiopoietin (Ang)-2, promote cellular sensitivity to pro-inflammatory factors such as vascular cell-adhesion molecule (VCAM)-1, and contribute to the progression of diabetes-related vascular disease ( 46 ). AGEs are non-enzymatic glycosylated forms of free amino acids that result from the interaction of glucose with lipids or proteins, bind to receptors to promote inflammation and oxidative stress, and are important in contributing to glomerulosclerosis and mesangial hypertrophy in DKD ( 47 – 49 ). On the other hand, ROS acts as an agonist of NF-κB signaling pathway to initiate the activation of downstream inflammatory signaling pathways and promotes the release of inflammatory factors such as IL-1β, TNF-α, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1) ( 50 – 54 ). We know that mitochondria are the main site of ROS production and a target organelle for oxidative stress ( 55 ). The ETC is the central component of the mitochondria for functional operation, and the sequential transfer of electrons through the ETC to complex IV creates an electrochemical proton gradient that drives the F 1 F 0 ATP synthase to produce ATP for OXPHOS ( 56 ). In addition, mitochondrial ROS production is increased and morphology is altered in high blood glucose environment of DM. Further, prolonged high glucose stimulation results in more electron donors such as NADH and FADH 2 being produced in the tricarboxylic acid (TCA) cycle, and too many electron donors entering the ETC, leading to a maximum mitochondrial voltage gradient ( 57 ). Uncoupling proteins (UCPs) reduce ROS production by dissipating the proton motive force and increasing the rate of elec

mtDNA is a double-stranded circular structure (consisting of light and heavy strands), localizes in the mitochondrial matrix, closing to IMM ( 82 ). The human mtDNA is 16,569 bp in length and consists of 37 genes, 22 tRNAs, 2 rRNAs and a non-coding region displacement-loop (D-loop) ( 83 , 84 ). However, the non-coding region controls the transcription and translation of mitochondrial proteins, but the high sequence mutagenicity in this region makes the mtDNA mutation rate approximately 10-20 times higher than nuclear DNA, with relevance to diseases such as aging and cancer ( 85 – 87 ). Moreover, due to the tight arrangement of genes in the ring structure of mtDNA, some genes overlap, and lack of histone protection, it is vulnerable to ROS generated by oxidative stress process, resulting in persistent damage to mtDNA ( 88 , 89 ). Mitochondria have a different DNA genetic system from nuclear DNA. mtDNA is responsible for encoding some of the mitochondrial proteins (such as the protein complexes that make up the ETC) and involved in mitochondrial biogenesis and signaling, with semi-autonomous genetic characteristics ( 82 , 90 , 91 ). Therefore, mtDNA is important for OXPHOS. Mutations in mtDNA and epigenetic changes can lead to blocked electron transport in the ETC, reducing ATP synthesis and promoting apoptosis. Further, transcription and packaging factor (TFAM), and transcription elongation factor (TEFM), essential cofactors for mtDNA replication and transcription, play an important role in the assembly and distribution of mtDNA-protein complexes and are thought to alleviate insulin resistance-induced oxidative stress ( 90 , 92 – 94 ). In addition, the organism equips mtDNA with DNA repair systems, and base excision repair (BER) is considered to be the main repair mechanism ( 95 ). BER maintains the normal structure of mtDNA by eliminating base mismatches caused by methylation, oxidation and alkylation, and by cleaving, gap-filling and connecting the structure of mtDNA ( 96 ). In addition, mismatch repair (MMR), homologous recombination (HR) and non-homologous end joining (NHEJ) are also important repair pathways of mtDNA ( 97 ). It has been documented that base mismatches caused by elevated ROS can be recognized and excised by the BER pathway to maintain the functional and structural integrity of mtDNA ( 98 – 100 ). Similarly, in vitro and in vivo studies of the MMR pathway in high glucose environments have shown that high glucose environments induce damage to mtDNA and also impair the repair of the MMR pathway ( 101 ) ( Figure 3 ). Figure 3 Excessive ROS damages mtDNA. ROS drives damage to mtDNA and the mtDNA repair systems, yet the damage of mtDNA repair systems is also an important contributor to mtDNA damage, followed by damage to mitochondrial protein-related transcription and synthesis pathways, ultimately leading to a vicious cycle of ETC abnormalities and subsequent the excessive generation of ROS. ROS, reactive oxygen species; mtDNA, mitochondrial DNA; ETC, electron transfer chain.

In a high glucose-induced retinal ganglion cells (RGC) model, Hesperidin (Citrus Flavonone) restored mitochondrial function, prevented loss of MMP and cyt-c release into the cytoplasm, prevented ROS production, increased intracellular levels of antioxidant enzymes and inhibited apoptosis ( 147 ). A study on metabolic memory of mitochondrial oxidative damage found that in primary rat retinal endothelial cells (rRECs) cultured in high glucose, MMP and cyt-c levels decreased and ROS levels increased in the model group compared to the control group as the duration of high glucose culture increased, suggesting that metabolic memory of mitochondrial oxidative damage can lead to DR ( 148 ). Another study on Berberine (BBR) showed that BBR alleviated oxidative stress (inhibited cyt-c leakage and ROS production and increased antioxidant enzyme levels) in diabetic rats and high glucose-induced Müller cells by inhibiting the NF-κB signaling pathway, thereby preventing DR ( 149 ). Leakage of cyt-c and increased accumulation of Bax in mitochondria in STZ-induced diabetic rats and high glucose cultured retinal endothelial cells and pericytes, which were inhibited by SOD and its mimics ( 150 ). In another study, in addition to demonstrating the protective effect of manganese superoxide dismutase (MnSOD) on the retina, it was also demonstrated that complex III might be a more significant source of superoxide compared to complex I ( 151 ). It had been suggested that MTP-131 (a novel mitochondrial targeting peptide) alleviated H 2 O 2 -induced oxidative stress in RGC-5 (blocking MMP depolarization and cyt-c release, reducing ROS production and preventing apoptosis) ( 152 ). NaHS (donor of H 2 S) blocked retinal abnormalities in diabetic rats and alleviated DR by inhibiting mitochondrial dysfunction and NF-κB activation ( 153 ). In high glucose-induced and platelet-derived growth factor-induced retinal pigment epithelial cells, researchers found that SIRT3 knockdown led to epithelial-mesenchymal transition and migration of epithelial cells, which was alleviated by overexpression of SIRT3. Further studies revealed that the cause was knockdown of SIRT3 leading to overproduction of mitochondrial ROS, suggesting the role for SIRT3 in inhibiting mitochondrial oxidative stress ( 154 ). In a vitro study, 670 nm photobiomodulation reduced high glucose-induced ROS production and maintained mitochondrial integrity in rat Müller glial cells ( 155 ). In an STZ-induced mouse experiment, STZ induced an increase in cytoplasmic and mitochondrial ROS, accompanied by lipid peroxidation and apoptosis, and a decrease in GSH and GSH-Px as well as optic nerve activity and vitamin A levels, which could be reversed by selenium and resveratrol ( 156 ). Some scientists investigated the mechanism of oxidative stress induced by high glucose in RGC-5, and concluded that high glucose induced ROS production, disrupted mitochondrial mechanisms (MMP, mtDNA and mitochondrial mass damage) and antioxidant mechanisms, and triggered the production of downstream inflammatory factors and neurodegenerative markers ( 157 ). A study on green tea (Camellia sinensis) and antioxidant vitamins showed that green tea and vitamins reduced retinal superoxide production and that green tea improved inhibition of ETC and complex III activity, but promoted tissue collagen matrix glyco-oxidation ( 150 ).

4.2.1 DKD Increased urinary 8-OHdG was detected in DKD-sensitive DBA/2J mice and human DKD specimens and showed a correlation between glomerular endothelin-1 receptor type A expression and increased mtDNA damage ( 166 ). The combination of dietary fenugreek (Trigonella foenum-graecum) seeds and onion (Allium cepa) was effective in reducing STZ-induced oxidative stress, lowering triglyceride and total cholesterol levels, reducing 8-OHdG and DNA fragmentation, and eliminating mtDNA deletions ( 167 ). In addition, salidroside alleviated renal fibrosis and kidney damage in DKD mice, and promoted the increase of mtDNA copy number and mitochondrial biogenesis ( 168 ).

A study comparing differences in mtDNA and transcript levels between diabetic and PGC-1α (-/-) diabetic mice found that PGC-1α (-/-) exacerbated neurological abnormalities in diseased mice, promoted mtDNA damage and protein oxidation, and led to more severe mitochondrial degeneration, demonstrating that modulation of PGC-1α may be a strategy for treating DPN ( 173 ). TFAM overexpression upregulated mtDNA and total TFAM levels, prevented the reduction of mtDNA copy number and inhibited motor and sensory nerve conduction abnormalities in diseased mice ( 174 ). A study on the neurological evaluation of 125 Italian T2DM patients noted that mtDNA was reduced in T2DM patients, this result was more significant in DPN patients and was associated with the MIR499A gene polymorphism ( 175 ).

Exendin-4 (a glucagon-like protein) increased GSH and magnesium superoxide dismutase levels, decreased NADPH oxidase levels, inhibited ROS production and cyt-c release, and prevented apoptosis in high glucose-induced adult human retinal pigment epithelial-19 cells by inhibiting p66Shc expression and activation ( 186 ). In a study on the relationship between retinal neuronal apoptosis and MnSOD in diabetic rats, it was noted that apoptosis increased in diabetic rats at 8 and 12 weeks, and the number of RGC cells decreased at 12 weeks, while MnSOD activity and mRNA levels decreased at 4, 8 and 12 weeks, indicating a close relationship between MnSOD and RGC apoptosis ( 187 ). Similarly, two other studies had shown that MnSOD overexpression inhibited the increase in 8-OHdG and nitrotyrosine levels, prevented the decrease in GSH and total antioxidant capacity caused by DR ( 188 , 189 ). An interesting study explored the response of knockdown of the Sigma 1 receptor (σ1RKO) on primary retinal Müller glial cells, showing that SOD1, CAT and GPX1 expression and protein levels were reduced in the σ1RKO group, as well as GSH and GSH/GSSG ratios, demonstrating that the neuroprotective effects of σ1R are related to the inhibition of oxidative stress ( 190 ).

High glucose-induced activation of the AGE, PKC, polyol and hexosamine pathways, as well as the formation of ROS in the mitochondria and cytoplasm, contribute to increased ROS production, and promote mitochondrial dysfunction and induce oxidative stress, mediating cellular dysfunction and accelerating the disease process ( 32 , 193 – 197 ) ( Figure 4 ). We have previously addressed the formation of ROS in the cytoplasm and mitochondria, so the following section focuses on the other four metabolic pathways. Figure 4 Abnormal metabolic pathways caused by hyperglycaemia. Hyperglycemia contributes to ROS production through the AGEs pathway, hexosamine pathway, PKC pathway, and polyol pathway; meanwhile, mitochondria and cytoplasm are also important sites for ROS production, which ultimately leads to oxidative stress. At the same time, oxidative stress can contribute to ETC abnormalities, reduce MMP, damage the mtDNA repair system and promote apoptosis. AGEs, advanced glycosylation end products; RAGE, the receptor for AGEs; NF-ΚB, nuclear factor kappa-light-chain-enhancer of activated B cells; AR, aldose reductase; SDH, sorbitol dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate; GSH, reduced glutathione; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAG, diacylglycerol; PKC, protein kinase C; VEGF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor-1; TGF-Β1, transforming growth factor; ROS, reactive oxygen species; ETC, electron transfer chain; mtDNA, mitochondrial DNA; NOX, NADPH oxidases; NADP + , nicotinamide adenine dinucleotide phosphate oxidized; O 2 .- , superoxide anion; SOD, superoxide dismutase; H 2 O 2 , hydrogen peroxide; •OH: hydroxyl radical. 5.1 AGEs/RAGE pathway Non-enzymatic glycosylation of proteins and other macromolecules caused by prolonged high glucose levels, resulting in a series of dehydration and fracture reactions leading to the production of AGEs, resulting in abnormal protein structure and function, and consequently abnormal physiological function ( 198 ). AGEs promote oxidative stress by impairing the ETC to promote ROS formation ( 199 ). At the same time, the production of ROS can in turn stimulate the production of AGEs, thus creating a vicious circle ( 200 ). In addition, AGEs mediate the activation of downstream inflammatory and fibrotic signaling pathways by binding to cell surface receptors (RAGE) ( 201 – 203 ).

High glucose promotes increased glycolysis, leading to greater diacylglycerol (DAG) production, while inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) increases DAG activity and activates the PKC pathway ( 210 ). Activation of the PKC pathway is often accompanied by increased production of inflammatory factors and vascular endothelial growth factor (VEGF), and is closely associated with the development of diabetic complications ( 193 , 211 – 215 ).

Oxidative stress is an imbalance in the redox state of the body, where excessive production of free radicals or damage to the antioxidant system, leads to a pathological outcome that is closely linked to the development of diseases such as cancer and metabolic disorders ( 219 ). ROS is a major component of free radicals, mainly produced in small amounts during OXPHOS in mitochondria, and plays an important role in cell signaling, cell proliferation and antibacterial immunity ( 220 , 221 ). However, the prolonged and persistent hyperglycaemic state of DM leads to an increase in cellular respiratory substrates entering the mitochondria, with excess electron donors impairing ETC, contributing to ROS production, mediating the breakdown of the proton electrochemical gradient, impaired MMP, increased cyt-c leakage and inadequate ATP synthesis ( 56 ). Due to the lack of histone protection of mtDNA, the high mutability of the non-coding region and the restriction of its loop structure, ROS can further damage mtDNA, leading to a reduction in the copy number of mtDNA, damage the genes responsible for encoding some mitochondrial proteins and impair the function of mitochondria ( 87 , 222 , 223 ). At the same time, the increased ROS can damage the repair system of mtDNA, further deepening the damage to mtDNA and causing functional impairment of mitochondria ( 224 , 225 ). In addition, the instability of ROS encourages cross-linking with macromolecular proteins, DNA and lipids, altering the structure and function of macromolecules and having toxic effects, further affecting cell function ( 226 ). However, ROS can also mediate the activation of downstream signaling pathways such as inflammation and fibrosis, leading to the progression of diabetic microvascular complications such as DKD and DPN ( 227 – 229 ). Studies have shown that some herbal active ingredients (puerarin, polydatin, quercetin, etc.), vitamin C, vitamin E, α-lipoic acid are important antioxidant strategies ( 160 , 163 , 164 , 230 – 233 ). Targeting mitochondria to overexpress catalase in mice extends lifespan and alleviates oxidative stress in diseases such as metabolic syndrome ( 234 ). A clinical trial of the drug elamipretide (a mitochondrial tetrapeptide that interacts with cardiolipin) showed that elamipretide significantly improved clinical symptoms and skeletal muscle performance in Barth syndrome ( 235 ). In addition, animal models have demonstrated that enzymatic antioxidants mimics (SOD mimics, GPX mimics and CAT mimics) can scavenge superoxide and inhibit oxidative stress ( 236 – 238 ). Recent studies have shown that bioadhesive hydrogel can promote oral wound healing in DM rats; novel nanoparticle can accelerate wound healing in DM and is an emerging and effective treatment strategy ( 239 , 240 ). Some combinations of antioxidants have also been shown to have antioxidant effects ( 241 – 243 ). The relevant literatures state that the combinations of ferulic acid and metformin have been shown to improve DM ( 244 ). And the combinations of superoxide dismutase, α-lipoic acid, acetyl-L-carnitine, and vitamin B 12 have been shown to improve sural nerve conduction velocity, amplitude and pain in patients with DPN ( 233 ). Therefore, antioxidants play a positive therapeutic role in the treatment of DM. However, many antioxidants suffer from poor solubility, unstable storage and gastrointestinal degradation, thus limiting the use of oxidants in clinical practice ( 245 ). In recent years antioxidant drugs have mainly focused on animal studies and have not been adequately tested in clinical trials, therefore, poorly supported by clinical data. The few drugs that have been clinically studied have not yielded satisfactory results either, and achieving effective drug concentrations in the body is an important issue for modern science. In addition to this, mtDNA, a key structure involved in oxidative stress, is under-researched for drugs targeting mtDNA, leading to a lack of development of antioxidant drugs. We hope to be able to characterize mitochondrial dysfunction in the high glucose state and look forward to providing a bit of new ideas for future experimental studies.

ZZ is responsible for writing and the conception of the article, QH is responsible for drawing and document sorting, and DZ is responsible for sorting out the ideas of the article. FL, XL, and WQ are responsible for controlling the overall quality of the article and revising the article. All authors contributed to the article and approved the submitted version.

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