Nrf2 protects against renal fibrosis induced by chronic cadmium exposure in mice.

Environmental cadmium (Cd) exposure is a serious public health concern, as the kidney is the primary target for Cd exposure. The present study aimed to investigate the role and underlying mechanisms of nuclear factor erythroid-derived 2-like 2 (Nrf2) in renal fibrosis induced by chronic Cd exposure. Nrf2 knockout (Nrf2-KO) mice and their wild-type littermates (Nrf2-WT) were exposed to 100 or 200 ppm Cd in drinking water for up to 16 or 24 weeks. Following the Cd exposures, Nrf2-KO mice showed elevated urinary neutrophil gelatinase-associated lipocalin (NGAL) and BUN levels compared to Nrf2-WT mice. Masson's trichrome staining and expression of fibrosis-associated proteins revealed that more severe renal fibrosis occurred in Nrf2-KO than that in Nrf2-WT mice. Renal Cd content in the Nrf2-KO mice exposed to 200 ppm Cd was lower than that in Nrf2-WT mice, which might be a consequence of the severe renal fibrosis in the Nrf2-KO mice. Mechanistic studies showed that Nrf2-KO mice exhibited higher levels of oxidative damage, lower antioxidant levels, and more regulated cell death, apoptosis in particular, than those in Nrf2-WT mice caused by Cd exposure. In conclusion, Nrf2-KO mice were more prone to develop renal fibrosis induced by chronic Cd exposure, partially due to a weakened antioxidant, detoxification capacity and increased oxidative damage.

Coexistence of cryoglobulinemia and ANCA-associated vasculitis in a chronic brucellosis patient -a case report and literature review.

BACKGROUND: The renal involvement of brucellosis is not common. Here we reported a rare case of chronic brucellosis accompanied by nephritic syndrome, acute kidney injury, the coexistence of cryoglobulinemia and antineutrophil cytoplasmic autoantibodies (ANCA) associated vasculitis (AAV) superimposed on iliac aortic stent implantation. The diagnosis and treatment of the case are instructive. CASE PRESENTATION: A 49-year-old man with hypertension and iliac aortic stent implantation was admitted for unexplained renal failure with signs of nephritic syndrome, congestive heart failure, moderate anemia and livedoid change in the left sole with pain. His past history included chronic brucellosis and he just underwent the recurrence and completed the 6 weeks of antibiotics treatment. He demonstrated positive cytoplasmic/proteinase 3 ANCA, mixed type cryoglobulinemia and decreased C3. The kidney biopsy revealed endocapillary proliferative glomerulonephritis with a small amount of crescent formation. Immunofluorescence staining revealed only C3-positive staining. In accordance with clinical and laboratory findings, post-infective acute glomerulonephritis superimposed with AAV was diagnosed. The patient was treated with corticosteroids and antibiotics and sustained alleviation of renal function and brucellosis was achieved during the course of a 3-month follow-up. CONCLUSIONS: Here we describe the diagnostic and treatment challenge in a patient with chronic brucellosis related glomerulonephritis accompanied by the coexistence of AAV and cryoglobulinemia. Renal biopsy confirmed the diagnosis of postinfectious acute glomerulonephritis overlapping with ANCA related crescentic glomerulonephritis, which was not ever reported in the literature. The patient showed a good response to steroid treatment which indicated the immunity-induced kidney injury. Meanwhile, it is essential to recognize and actively treat the coexisting brucellosis even when there are no clinical signs of the active stage of infection. This is the critical point for a salutary patient outcome for brucellosis associated renal complications.

LNA-anti-miR-150 alleviates renal interstitial fibrosis by reducing pro-inflammatory M1/M2 macrophage polarization.

Renal interstitial fibrosis (RIF) is a common pathological feature contributing to chronic injury and maladaptive repair following acute kidney injury. Currently, there is no effective therapy for RIF. We have reported that locked nuclear acid (LNA)-anti-miR-150 antagonizes pro-fibrotic pathways in human renal tubular cells by regulating the suppressor of cytokine signal 1 (SOCS1)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. In the present study, we aimed to clarify whether LNA-anti-miR-150 attenuates folic acid-induced RIF mice by regulating this pathway and by reducing pro-inflammatory M1/M2 macrophage polarization. We found that renal miR-150 was upregulated in folic acid-induced RIF mice at day 30 after injection. LNA-anti-miR-150 alleviated the degree of RIF, as shown by periodic acid-Schiff and Masson staining and by the expression of pro-fibrotic proteins, including alpha-smooth muscle actin and fibronectin. In RIF mice, SOCS1 was downregulated, and p-JAK1 and p-STAT1 were upregulated. LNA-anti-miR-150 reversed the changes in renal SOCS1, p-JAK1, and p-STAT1 expression. In addition, renal infiltration of total macrophages, pro-inflammatory M1 and M2 macrophages as well as their secreted cytokines were increased in RIF mice compared to control mice. Importantly, in folic acid-induced RIF mice, LNA-anti-miR-150 attenuated the renal infiltration of total macrophages and pro-inflammatory subsets, including M1 macrophages expressing CD11c and M2 macrophages expressing CD206. We conclude that the anti-renal fibrotic role of LNA-anti-miR-150 in folic acid-induced RIF mice may be mediated by reducing pro-inflammatory M1 and M2 macrophage polarization via the SOCS1/JAK1/STAT1 pathway.

Co-occurrence of IgA nephropathy and IgG4-Tubulointersitial nephritis effectively treated with tacrolimus: a case report.

BACKGROUND: Cases of concurrent immunoglobulin A nephropathy (IgAN) and IgG4-related tubulointerstitial nephritis (IgG4-TIN) are rare and previous case reports have lacked important data. KDIGO suggests a treatment with systemic glucocorticoids in IgAN patients. Glucocorticoids are recommended as the first-line therapy for IgG4-TIN. The use of tacrolimus as a long-term maintenance treatment has not been described. We report the case of a man who developed IgAN and IgG4-TIN without abnormalities in extra-renal tissue, without renal function abnormalities or impairment as well, and was treated by tacrolimus as a long-term maintenance during 45 months follow-up. CASE PRESENTATION: A 56-year-old Chinese man first presented to our hospital with the chief complaint of foamy urine for 1 year and hematuria for 3 months, with a medical history of hypertension. Testing revealed a notable increase in serum IgG4 level without abnormalities in renal function or imaging, or in dysfunction other organs. Renal biopsy showed mesangial extracellular matrix proliferation, increased mesangial cell numbers and infiltration of plasma cells. Immunofluorescence showed mesangial positivity for IgA and C3. Immunohistochemistry staining showed widespread IgG4 and increased CD38 and CD138 expression. Electron microscopy showed immune complexes located on the tubular basement membrane. He was diagnosed with IgAN and IgG4-TIN. He received glucocorticoids, leflunomide and tacrolimus to induce remission. He was given tacrolimus as long-term maintenance treatment. When tacrolimus was temporarily withdrawn, proteinuria recurred. After resuming tacrolimus therapy, he again entered complete remission. After 45 months of therapy, he remains in complete remission and the serum IgG4 level is normal. CONCLUSIONS: The finding of concurrent IgAN and IgG4-TIN without abnormalities in renal function, imaging or extra-renal tissue is rare and their coexistence may be coincidental. Long-term treatment with tacrolimus proved effective and he has remained in remission during 45 months follow-up.

Nrf2 deficiency aggravates the kidney injury induced by subacute cadmium exposure in mice.

Cadmium (Cd) is a heavy metal pollutant that adversely effects the kidney. Oxidative stress and inflammation are likely major mechanisms of Cd-induced kidney injury. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is crucial in regulating antioxidant and inflammatory responses. To investigate the role of Nrf2 in the development of subacute Cd-induced renal injury, we utilized Nrf2 knockout (Nrf2-KO) and control mice (Nrf2-WT) which were given cadmium chloride (CdCl2, 1 or 2 mg/kg i.p.) once daily for 7 days. While subacute CdCl2 exposure induced kidney injury in a dose-dependent manner, after the higher Cd dosage exposure, Nrf2-KO mice showed elevated blood urea nitrogen (BUN) and urinary neutrophil gelatinase-associated lipocalin (NGAL) levels compared to control. In line with the findings, the renal tubule injury caused by 2 mg Cd/kg, but not lower dosage, in Nrf2-KO mice determined by Periodic acid-Schiff staining was more serious than that in control mice. Further mechanistic studies showed that Nrf2-KO mice had more apoptotic cells and severe oxidative stress and inflammation in the renal tubules in response to Cd exposures. Although there were no significant differences in Cd contents of tissues between Cd-exposed Nrf2-WT and Nrf2-KO mice, the mRNA expression of Nrf2 downstream genes, including heme oxygenase 1 and metallothionein 1, were significantly less induced by Cd exposures in the kidney of Nrf2-KO compared with Nrf2-WT mice. In conclusion, Nrf2-deficient mice are more sensitive to kidney injury induced by subacute Cd exposure due to a muted antioxidant response, as well as a likely diminished production of specific Cd detoxification metallothioneins.

circHIPK3 Exacerbates Folic Acid-Induced Renal Tubulointerstitial Fibrosis by Sponging miR-30a.

Renal tubulointerstitial fibrosis is a common pathological feature of progressive chronic kidney disease (CKD), and current treatment has limited efficacy. The circular RNA circHIPK3 is reported to participate in the pathogenesis of various human diseases. However, the role of circHIPK3 in renal fibrosis has not been examined. In this study, we aimed to determine whether and how circHIPK3 might participate in the pathogenesis of renal fibrosis. Mice received a peritoneal injection of folic acid (250 mg/kg). Of note, 30 days later, renal fibrosis was present on periodic acid-Schiff (PAS) and Masson staining, and mRNA and protein of profibrotic genes encoding fibronectin (FN) and collagen 1 (COL1) were increased. Renal circHIPK3 was upregulated, while miR-30a was downregulated, assessed by quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). The expression of transforming growth factor beta-1 (TGF-ß1) was increased by qPCR analysis, immunoblotting, and immunofluorescence. Renal circHIPK3 negatively correlated with miR-30a, and kidney miR-30a negatively correlated with TGF-ß1. Target Scan and miRanda algorithms predicted three perfect binding sites between circHIPK3 and miR-30a. We found that circHIPK3, miR-30a, and TGF-ß1 colocalized in the cytoplasm of human tubular epithelial cells (HK-2 cells) on FISH and immunofluorescence staining. We transfected circHIPK3 and a scrambled RNA into HK-2 cells; miR-30a was downregulated, and the profibrotic genes such as TGF-ß1, FN, and COL1 were upregulated and assessed by qPCR, immunoblotting, and immunofluorescence staining. Third, the upregulation of circHIPK3, downregulation of miR-30a, and overproduction of profibrotic FN and COL1 were also observed in HK-2 cells exposed to TGF-ß1. Finally, renal biopsies from patients with chronic tubulointerstitial nephritis manifested similar expression patterns of circHIPK3, miR-30a, and profibrotic proteins, such as TGF-ß1, FN, and COL1 as observed in the experimental model. A feed-forward cycle was observed among circHIPK3, miR-30a, and TGF-ß1. Our results suggest that circHIPK3 may contribute to progressive renal fibrosis by sponging miR-30a. circHIPK3 may be a novel therapeutic target for slowing CKD progression.

miR-150-Based RNA Interference Attenuates Tubulointerstitial Fibrosis through the SOCS1/JAK/STAT Pathway In Vivo and In Vitro.

We investigated whether microRNA-150 (miR-150)-based RNA interference (RNAi) ameliorates tubular injury and tubulointerstitial fibrosis. Mice injected with folic acid developed tubulointerstitial fibrosis at day 30. miR-150 levels were increased at day 7 and peaked at day 30. At day 30, protein levels of α-smooth muscle actin, fibronectin (FN), and collagen 1 (COL-1) were increased, while suppressor of cytokine signal 1 (SOCS1) was decreased. Kidneys manifested increased macrophage numbers and increased expression of potential mediators: interferon-γ, interleukin-6, and tumor necrosis factor-α. Locked nucleic acid-anti-miR-150, started prior to or after tubular injury and administered twice weekly for 4 weeks, reversed renal inflammation and fibrosis. In HK-2 cells, co-culture with macrophages increased miR-150 expression and decreased SOCS1. Janus kinase (JAK) and signal transducer and activators of transcription (STAT) pathway-related proteins p-JAK1, p-JAK2, p-STAT1, p-STAT3, and pro-fibrotic genes encoding α-smooth muscle actin, FN, and COL-1 were all upregulated. The miR-150 antagonist reversed these transcriptional changes. Lastly, in renal biopsies from patients with chronic interstitial fibrosis, renal miR-150, and pro-fibrotic gene expression and macrophage numbers were increased, while SOCS1 expression was decreased. In conclusion, miR-150-based RNAi is as a potential novel therapeutic agent for tubulointerstitial fibrosis, suppressing the SOCS1/JAK/STAT pathway and reducing macrophage influx.

CircZNF609 is involved in the pathogenesis of focal segmental glomerulosclerosis by sponging miR-615-5p.

As a set of distinct syndromes, focal segmental glomerulosclerosis (FSGS) is the most common cause of adult nephrotic syndrome with diverse mechanisms. We recently found that expression of the circular RNA circZNF609 is increased in renal biopsies of lupus nephritis patients. In the present study, we aimed to determine whether circZNF609 participates in the pathogenesis of FSGS in mice given Adriamycin. In FSGS mice, circZNF609 was upregulated while miR-615-5p was downregulated in FSGS mice analyzed by qPCR and fluorescence in situ hybridization (FISH). Expression of podocyte proteins Wilms tumor 1 (WT1) and podocin were decreased, while expression of collagen 1 (COL1) and transforming growth factor-beta1 (TGF-ß1) were increased on Western blotting. Renal circZNF609 levels were positively correlated and miR-615-5p levels were negatively correlated with the degree of podocyte injury and renal fibrosis. Importantly, circZNF609 and miR-615-5p co-localized to glomeruli and tubules on FISH. Perfect match seeds were found between circZNF609 and miR-615-5p and COL1 mRNA, leading us to explore mechanisms of circZNF609 in bovine serum albumin (BSA) stimulating HK-2 cells, which model the toxicity of proteinuria on tubular cells. In vitro studies, circZNF609 increased and miR-615-5p decreased after BSA treatment and were negatively correlated with each other. COL1 and TGF-ß1 were both upregulated and negatively correlated with miR-615-5p. Lastly, circZNF609 expression increased in glomeruli and tubules of FSGS patient renal biopsies. We conclude that circZNF609 may play an important role in FSGS by sponging miR-615-5p.

LNA-anti-miR-150 ameliorated kidney injury of lupus nephritis by inhibiting renal fibrosis and macrophage infiltration.

BACKGROUND: The prevalence of lupus nephritis (LN) remains high despite various emerging monoclonal antibodies against with targeting systemic lupus erythematosus (SLE). Renal fibrosis is the main feature of late stage LN, and novel therapeutic agents are still needed. We previously reported that microRNA (miR)-150 increases in renal biopsies of American LN patients and that miR-150 agonist promotes fibrosis in cultured kidney cells. Presently, we aim to verify whether locked nucleic acid (LNA)-anti-miR-150 can ameliorate LN in mice and to investigate its corresponding mechanisms. METHODS: We first observed natural history and renal miR-150 expression in female Fcgr2b-/- mice of a spontaneously developed LN model. We then verified miR-150 renal absorption and determined the dose of the suppressed miR-150 by subcutaneous injection of LNA-anti-miR-150 (2 and 4 mg/kg). Thirdly, we investigated the therapeutic effects of LNA-anti-miR-150 (2 mg/kg for 8 weeks) on LN mice and the corresponding mechanisms by studying fibrosis-related genes, cytokines, and kidney resident macrophages. Lastly, we detected the expression of renal miR-150 and the mechanism-associated factors in renal biopsies from new onset untreated LN patients. RESULTS: Fcgr2b-/- mice developed SLE indicated by positive serum autoantibodies at age 19 weeks and LN demonstrated by proteinuria at age 32 weeks. Renal miR-150 was overexpressed in LN mice compared to wild type mice. FAM-labeled LNA-anti-miR-150 was absorbed by both glomeruli and renal tubules. LNA-anti-miR-150 suppressed the elevated renal miR-150 levels in LN mice compared to the scrambled LNA without systemic toxicity. Meanwhile, serum double strand-DNA antibody, proteinuria, and kidney injury were ameliorated. Importantly, the elevated renal pro-fibrotic genes (transforming growth factor-ß1, α-smooth muscle antibody, and fibronectin) and decreased anti-fibrotic gene suppressor of cytokine signal 1 were both reversed. Renal pro-inflammatory cytokines (interferon-γ, interleukin-6, and tumor necrosis factor-α) and macrophages were also decreased. In addition, the changes of renal miR-150 and associated proteins shown in LN mice were also seen in human subjects. CONCLUSIONS: LNA-anti-miR-150 may be a promising novel therapeutic agent for LN in addition to the current emerging monoclonal antibodies, and its renal protective mechanism may be mediated by anti-fibrosis and anti-inflammation as well as reduction of the infiltrated kidney resident macrophages.

Clinical significance and expression of serum Golgi protein 73 in primary hepatocellular carcinoma.

BACKGROUND: Golgi protein 73 (GP73) is a potential serum marker for the diagnosis of hepatocellular carcinoma (HCC). However, the diagnostic value of GP73 for HCC remains controversy, and little is known about the relationship between serum GP73 concentration and clinical characteristics of HCC. This study was designed to analyze the clinical values of GP73 in diagnosing HCC and the relationship between GP73 level and clinical characteristics in HCC patients. MATERIALS AND METHODS: In this study, a total of 443 serum samples including 180 patients with HCC, 61 patients with liver cirrhosis (LC), 99 chronic hepatitis patients, and 103 healthy individuals were enrolled from November 2011 to April 2013. Enzyme-linked immunosorbent assay and chemiluminescent immunoassay were used to detect the serum level of GP73 and other biomarkers. RESULTS: GP73 level in HCC group was significantly higher than LC group, chronic hepatitis B group, and healthy control group. In HCC group, GP73 level significantly increased in patients with lymphatic metastasis; moreover, GP73 level in Child-Pugh Class B and C groups was statistically significantly higher than that in Child-Pugh Class A group (P < 0.05). The area under the receiver operating characteristic (AUROC) curve of GP73 and alpha-fetoprotein (AFP) was 0.840 and 0.718, respectively, when diagnosing HCC. Moreover, the AUROC curve by use of the combination of GP73 and AFP was 0.903. The differences among these three aspects were statistically significant (P < 0.05). CONCLUSION: GP73 was better than AFP for the diagnosis of HCC, and the expression of serum GP73 is related with the clinical characteristics of HCC patients.

circHLA-C Plays an Important Role in Lupus Nephritis by Sponging miR-150.

Circular RNAs (circRNAs) participate in the pathogenesis of various diseases by sponging microRNAs (miRs). However, the roles of circRNAs remain unreported in glomerular diseases. We previously reported that miR-150 positively correlated with renal chronicity index in patients with lupus nephritis (LN). We aimed to investigate renal circRNA profiling and the interaction between circRNAs and miR-150 in LN patients. Six renal biopsies from untreated female patients with LN class IV and five normal kidney tissues from urology patients were used for circRNA sequencing. 171 circRNAs with 2-fold differential expression were identified in LN compared with normal control. Ten selected circRNAs were validated by real-time qPCR, and seven circRNAs showed the same significant increases as the sequencing results. circHLA-C positively correlated with proteinuria (R = 0.92, p < 0.01), serum creatinine (R = 0.76, p = 0.08), renal activity index (R = 0.88, p < 0.05), and crescentic glomeruli (R = 0.93, p < 0.01). Renal circHLA-C increased 2.72-fold, and miR-150 decreased 66% in LN compared with normal control (p < 0.05). Bio-informatic analysis predicted miR-150 was regulated by circHLA-C and displayed one perfect match seed between circHLA-C and miR-150. The renal miR-150 showed a tendency of negative correlation with circHLA-C in LN patients. In conclusion, circHLA-C may play an important role in the pathogenesis of lupus nephritis by sponging miR-150.

Nrf2 deficiency promotes the progression from acute tubular damage to chronic renal fibrosis following unilateral ureteral obstruction.

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a central mediator of cellular responses to oxidative stress. We hypothesized that Nrf2 modulates progression from acute tubular damage to renal fibrosis. We asked whether Nrf2 deletion increases renal injury in mice following unilateral ureteral obstruction (UUO). Methods: We explored the time course of renal injury and Nrf2 expression in Nrf2+/+ mice following UUO. We compared Nrf2+/+ and Nrf2-/- mice following UUO in tubular damage, transdifferentiation [vimentin, proliferating cell nuclear antigen (PCNA)], fibrosis [fibronectin, α-smooth muscle actin (SMA)], antioxidative and inflammatory responses. We studied Nrf2 in renal biopsies of patients with acute, subacute and chronic tubulointerstitial nephritis (TIN). Results: In Nrf2+/+ mice, renal Nrf2 expression and Nrf2-regulated glutamate-cysteine ligase catalytic (Gclc) and heme oxygenase-1 (Ho-1) were elevated, and renal injury occurred between 2 and 14 days after UUO. On Day 2 following UUO, in Nrf2-/- mice compared with Nrf2+/+ mice, tubular damage, apoptotic cell numbers, cleaved caspase3 and cleaved-poly ADP-ribose polymerase were increased. On Day 5, protein levels of vimentin and PCNA and the co-expressed cells of both proteins were increased. On Day 14, fibronectin and α-SMA protein levels were increased. Nrf2 deletion decreased expression of antioxidative genes (Gclc and Ho-1) and increased expression of inflammatory response genes (Tgfß, Tnf, IL-6, IL-1ß and F4/80). Finally, Nrf2 expression was upregulated in renal biopsies of patients with TIN. Conclusions: Following UUO, Nrf2 deficiency increased tubular damage, transdifferentiation, fibrosis and inflammatory response while decreasing antioxidative responses. The renal protective role of Nrf2 in the development of tubulointerstitial fibrosis in UUO may be mediated by antioxidative and anti-inflammatory pathways.

Elevated Serum Levels of Retinol-Binding Protein 4 Are Associated with Breast Cancer Risk: A Case-Control Study.

BACKGROUND: Retinol binding protein 4 (RBP4) is a recently identified adipokine that is elevated in patients with obesity or type 2 diabetes. A growing body of research has shown that RBP4 is associated with several types of cancer. However, no studies have investigated the relationship between serum RBP4 levels and breast cancer risk. We performed a case-control study to evaluate the association between serum RBP4 levels and the risk of breast cancer. METHODS: From August 2012 to December 2013, four-hundred subjects including 200 patients diagnosed with primary breast cancer and 200 matched healthy women were consecutively enrolled from Affiliated Hospital of Qingdao University Medical College. Blood samples were collected from healthy controls and breast cancer patients before commencement of treatment. Enzyme-linked immunosorbent assay was used to evaluate the serum RBP4 levels in separated serum samples. Meanwhile, the characteristics of breast cancer cases and controls were collected from medical records and pathological data. RESULTS: The serum levels of RBP4 were significantly higher in patients with breast cancer than that in the healthy control group (33.77±9.92 vs. 28.77±6.47µg/ml, P < 0.05). Compared to the subjects in the lowest quartile of serum RBP4 level, the adjusted ORs (95% CIs) is 2.16(1.01-4.61) and 2.07 (1.07-4.00) for women in the second and highest RBP4 tertile, respectively. For breast cancer patients, patients with PR or ER negative displayed significantly higher serum RBP4 levels than those with PR or ER positive. CONCLUSION: Our results for the first time suggested serum RBP4 levels could be associated with the risk of breast cancer. However, further prospective studies are essential to confirm these observed results.

Melatonin inhibits manganese-induced motor dysfunction and neuronal loss in mice: involvement of oxidative stress and dopaminergic neurodegeneration.

Excessive manganese (Mn) induces oxidative stress and dopaminergic neurodegeneration. However, the relationship between them during Mn neurotoxicity has not been clarified. The purpose of this study was to investigate the probable role of melatonin (MLT) against Mn-induced motor dysfunction and neuronal loss as a result of antagonizing oxidative stress and dopaminergic neurodegeneration. Mice were randomly divided into five groups as follows: control, MnCl2, low MLT + MnCl2, median MLT + MnCl2, and high MLT + MnCl2. Administration of MnCl2 (50 mg/kg) for 2 weeks significantly induced hypokinesis, dopaminergic neurons degeneration and loss, neuronal ultrastructural damage, and apoptosis in the substantia nigra and the striatum. These conditions were caused in part by the overproduction of reactive oxygen species, malondialdehyde accumulation, and dysfunction of the nonenzymatic (GSH) and enzymatic (GSH-Px, superoxide dismutase, quinone oxidoreductase 1, glutathione S-transferase, and glutathione reductase) antioxidative defense systems. Mn-induced neuron degeneration, astrocytes, and microglia activation contribute to the changes of oxidative stress markers. Dopamine (DA) depletion and downregulation of DA transporter and receptors were also found after Mn administration, this might also trigger motor dysfunction and neurons loss. Pretreatment with MLT prevented Mn-induced oxidative stress and dopaminergic neurodegeneration and inhibited the interaction between them. As a result, pretreatment with MLT significantly alleviated Mn-induced motor dysfunction and neuronal loss. In conclusion, Mn treatment resulted in motor dysfunction and neuronal loss, possibly involving an interaction between oxidative stress and dopaminergic neurodegeneration in the substantia nigra and the striatum. Pretreatment with MLT attenuated Mn-induced neurotoxicity by means of its antioxidant properties and promotion of the DA system.