Protective role of curcumin in cadmium-induced testicular injury in mice by attenuating oxidative stress via Nrf2/ARE pathway.

The aim of the present study was to investigate whether curcumin (CUR) can ameliorate cadmium-induced reproductive toxicity and its mechanism. A total of 48 male mice were equally divided into 4 groups: control, CdCl2 (2 mg/kg, intraperitoneally inject) curcumin (50 mg/kg, intraperitoneally inject), co-treatment with curcumin (50 mg/kg), and CdCl2 (2 mg/kg) for 10 days. The results demonstrated that CdCl2 reduces sperm motility, decreases the sperm density and serum testosterone content, and significantly improves the rate of sperm deformity. CdCl2 increased the level of testicular total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) activity, and glutathione (GSH), and CdCl2 declined the level of malondialdehyde (MDA). However, the semen quality of the mice in the curcumin intervention group was improved. Moreover, the testosterone content and antioxidant capacity were increased. In the Cd group mice, the expression of testicular Nrf2, as well as the mRNA and protein expressions of the downstream target molecules, glutathione peroxidase (GSH-Px), and γ-glutamylcysteine synthetase (γ-GCS) of Nrf2 declined, while the above genetic expressions elevated significantly in the curcumin intervention group. Our results suggested that curcumin could protect against Cd-induced testicular injury via activating the Nrf2/ARE signaling pathway.

Sulforaphane Protect Against Cadmium-Induced Oxidative Damage in mouse Leydigs Cells by Activating Nrf2/ARE Signaling Pathway.

Cadmium (Cd) is harmful for humans and animals, especially for the reproductive system. However, the mechanism of its toxicity has not been elucidated, and how to alleviate its toxicity is very important. This study aimed to explore the role and mechanism of action of sulforaphane (SFN) in protecting mouse Leydigs (TM3) cells from cadmium (Cd)-induced damage. The half-maximal inhibitory concentration (IC50) of Cd and the safe doses of SFN were determined using a methyl thiazolyl tetrazolium (MTT) assay. The testosterone secretion from TM3 cells was measured using the enzyme-linked immunosorbent assay. The intracellular oxidative stress was evaluated using corresponding kits. The cell apoptosis was detected using flow cytometry. The mRNA expression of genes associated with NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling was detected using reverse transcription⁻polymerase chain reaction, including Nrf2, heme oxygenase I (HO-1), glutathione peroxidase (GSH-Px), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). The protein expression of Nrf2, GSH-Px, HO-1, γ-GCS, and NQO1 was detected using Western blot analysis. The results showed that the IC50 of Cd to TM3 cells was 51.4 µmol/L. SFN reduced the release of lactate dehydrogenase from Cd-exposed cells. Cd + SFN 2.5 treatment significantly elevated testosterone concentration compared with the Cd group (p < 0.05). SFN significantly increased total superoxide dismutase (T-SOD) and GSH-Px activity and GSH content in Cd-treated cells (p < 0.05; p < 0.01), inhibited the production of malondialdehyde or reactive oxygen species caused by Cd (p < 0.05; p < 0.01), and reduced the apoptotic rate of Cd-induced TM3 cells (p < 0.01). SFN upregulated the mRNA expression of Nrf2, GSH-Px, HO-1, NQO1, and γ-GCS in Cd-treated cells, indicating the protective effect of SFN against Cd-induced oxidative stress or cell apoptosis by activating the Nrf2/ARE signaling pathway.

Protective Mechanism of Sulforaphane on Cadmium-Induced Sertoli Cell Injury in Mice Testis via Nrf2/ARE Signaling Pathway.

The present study evaluated the mechanism underlying the protective effect of sulforaphane (SFN) on cadmium (Cd)-induced Sertoli cell (TM4 cells) injury in mice. The apoptosis rate of cells in each group was detected by flow cytometry. It was determined the effect of SFN on the expression of downstream molecular targets of Nrf2/ARE axis and on the lipid peroxide content. The related genes involved in the nuclear factor E2-related factor 2(Nrf2)/antioxidant response element (ARE) signaling pathway were evaluated by RT-PCR; for example, the mRNA expression levels of Nrf2, heme oxygenase-1 (HO-1), glutathione peroxidase (GSH-Px), quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS), while the protein expression levels were assessed by Western blot. Our results showed that the mRNA and protein expression levels of Nrf2, HO-1, NQO1, GSH-Px, and γ-GCS were increased in various degree when the Sertoli cells were to added different concentrations of SFN. Our results also showed that SFN reduced the apoptosis rate, increased the activity of T-SOD, inhibited the increase of the MDA content caused by Cd. Meanwhile, SFN could increase the mRNA and protein expression levels of Nrf2, HO-1 and NQO1 and reduced the mRNA and protein expression levels of GSH-Px and γ-GCS caused by Cd in Sertoli cells (p < 0.01). Taken together, SFN could improve the antioxidant capacity of Sertoli cells, and exert a protective effect on the oxidative damage and apoptosis of Cd-induced Sertoli cells through the activation of Nrf2/ARE signal transduction pathway.

Protective effects of proanthocyanidins against cadmium-induced testicular injury through the modification of Nrf2-Keap1 signal path in rats.

The purpose of this study was to evaluate the potential chemoprotective effects of proanthocyanidins (PAs) against cadmium (Cd)-induced oxidative damage of testes via Nrf2-Keap1 signal pathway in rats. Briefly, by using biochemical histological analysis, as well as the real time PCR and western blot approach, oxidative damage in rat testicular tissue was observed after exposure to Cd. In addition, significant down-regulations of mRNA and protein levels of nuclear erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), γ-glutamyl cysteine synthetase (γ-GCS), glutathione peroxidase (GSH-Px) and quinone oxidoreductase 1 (NQO1), as well as a significant up-regulation of Kelch sample related protein-1 (Keap1) levels in testicular tissue were observed after Cd exposure. Notably, these alterations were reverted back to near normalcy in Cd+PAs group rats. In conclusion, PAs exhibited a significant chemopreventive potential against Cd-induced testicular oxidative damage in rats, possibly through the modification of Nrf2-Keap1 signal path.

Protective effect of proanthocyanidin on mice Sertoli cell apoptosis induced by zearalenone via the Nrf2/ARE signalling pathway.

This study evaluated the protective effect of proanthocyanidin (PC) on the cytotoxicity of the Sertoli cell TM4 of mice, as induced by zearalenone (ZEA). Flow cytometry was used to detect the apoptosis rate of cells in each group. The activities of antioxidant enzymes and the content of antioxidant substances were detected by using a proprietary kit; the RT-PCR method was used to detect the expression level of mRNA, the related genes of Nrf2/ARE signal pathway, the nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase (GSH-Px), quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase (γ-GCS) and the expression level of mRNA, the apoptosis-related genes, Bcl-2 and Bax; the Western-blot method was used to detect the protein expression levels of Nrf2, GSH-Px, HO-1, γ-GCS and NQO1 in each group. Our results showed that PC could reduce the apoptosis rate of the TM4 cells exposed to ZEA (p < 0.01); PC could enhance the decrease in the activities of T-SOD and GSH-Px induced by ZEA (p < 0.05), reduce the increase in the content of MDA, as caused by ZEA; PC could significantly up-regulate the down-regulation levels of the mRNA and protein of Nrf2, GSH-Px, HO-1, γ-GCS and NQO1 induced by ZEA. PC could enhance the decrease in the mRNA expression level of Bcl-2 and down-regulate the mRNA expression of Bax induced by ZEA (p < 0.05). These results demonstrated that PC conferred protective effects against oxidative damage and apoptosis of TM4 cells induced by ZEA. The protection mechanism of PC on TM4 cells might act through the activation of the Nrf2/ARE signalling pathway.

Sulforaphane Prevents Testicular Damage in Kunming Mice Exposed to Cadmium via Activation of Nrf2/ARE Signaling Pathways.

Sulforaphane (SFN) is a natural and highly effective antioxidant. Studies suggest that SFN protects cells and tissues against cadmium (Cd) toxicity. This study investigated the protective effect of SFN against oxidative damage in the testes of Kunming mice exposed to cadmium, and explored the possible molecular mechanisms involved. Cadmium greatly reduced the serum testosterone levels in mice, reduced sperm motility, total sperm count, and increased the sperm deformity rate. Cadmium also reduces superoxide dismutase (T-SOD) and glutathione (GSH) levels and increases malondialdehyde (MDA) concentrations. SFN intervention improved sperm quality, serum testosterone, and antioxidant levels. Both mRNA and protein expression of mouse testicular nuclear factor-erythroid 2-related factor 2 (Nrf2) was reduced in cadmium-treated group. Furthermore, the downstream genes of Nrf2, glutathione peroxidase (GSH-Px), γ-glutamyl cysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase-1 (NQO1) were also decreased in cadmium-treated group. SFN intervention increases the expression of these genes. Sulforaphane prevents cadmium-induced testicular damage, probably via activation of Nrf2/ARE signaling.

The Protective Effect of Grape-Seed Proanthocyanidin Extract on Oxidative Damage Induced by Zearalenone in Kunming Mice Liver.

Although grape-seed proanthocyanidin extract (GSPE) demonstrates strong anti-oxidant activity, little research has been done to clearly reveal the protective effects on the hepatotoxicity caused by zearalenone (ZEN). This study is to explore the protective effect of GSPE on ZEN-induced oxidative damage of liver in Kunming mice and the possible protective molecular mechanism of GSPE. The results indicated that GSPE could greatly reduce the ZEN-induced increase of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. GSPE also significantly decreased the content of MDA but enhanced the activities of antioxidant enzymes SOD and GSH-Px. The analysis indicated that ZEN decreased both mRNA expression levels and protein expression levels of nuclear erythroid2-related factor2 (Nrf2). Nrf2 is considered to be an essential antioxidative transcription factor, as downstream GSH-Px, γ-glutamyl cysteine synthetase (γ-GCS), hemeoxygenase-1 (HO-1), and quinone oxidoreductase 1 (NQO1) decreased simultaneously, whereas the pre-administration of GSPE groups was shown to elevate these expressions. The results indicated that GSPE exerted a protective effect on ZEN-induced hepatic injury and the mechanism might be related to the activation of the Nrf2/ARE signaling pathway.

Intervention of Grape Seed Proanthocyanidin Extract on the Subchronic Immune Injury in Mice Induced by Aflatoxin B1.

The aim was to investigate the prevention of grape seed proanthocyanidin extract (GSPE) on the subchronic immune injury induced by aflatoxin B1 (AFB1) and the possible ameliorating effect of GSPE in mice. The subchronic AFB1-induced immune injury mice model was set up with the continuous administration of 100 µg/kg body weight (BW) AFB1 for six weeks by intragastric administration. Then, intervention with different doses (50 and 100 mg/kg BW) of GSPE was conducted on mice to analyze the changes of body weight, immune organ index, antioxidant capability of spleen, serum immunoglobulin content, and the expression levels of inflammatory cytokines. The prevention of GSPE on the immune injury induced by AFB1 was studied. The GSPE could relieve the AFB1-induced reduction of body weight gain and the atrophy of the immune organ. The malondialdehyde (MDA) level of the spleen in the AFB1 model group significantly increased, but levels of catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-P(X)), and superoxide dismutase (SOD) significantly decreased. The GSPE could significantly inhibit the oxidative stress injury of the spleen induced by AFB1. AFB1 exposure could not significantly change the contents of IgA, IgG, or IgM. AFB1 significantly improved the expression of interleukin 1ß (IL-1ß), IL-6, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). Additionally, GSPE could decrease the expression of these four proinflammatory factors to different degrees and inhibit the inflammatory reaction of mice. The results suggest that GSPE alleviates AFB1-induced oxidative stress and significantly improves the immune injury of mice induced by AFB1.

Seroprevalence of Toxoplasma gondii infection in police dogs in Shenyang, Northeastern China.

In recent years, worldwide surveys of Toxoplasma gondii infection in dogs have been reported. However, only limited surveys of T. gondii infection in police dogs have been available, including China. In the present study, we report the seroprevalence of T. gondii in police dogs in Shenyang, northeastern China. Sera from 291 police dogs were examined for T. gondii antibodies with the modified agglutination test (MAT), and 30.9% animals were tested seropositive. The results of the present study indicated a relatively high prevalence of T. gondii infection in police dogs in Shenyang, China.

Seroprevalence of Toxoplasma gondii in slaughtered horses and donkeys in Liaoning province, northeastern China.

BACKGROUND: Toxoplasma gondii is an important zoonotic pathogen infecting humans and almost all warm-blooded animals. The most common sources of human infection are ingestion of tissue cysts in raw or undercooked meat. However, limited information is available about T. gondii infection in horses and donkeys in China. In the present study, we report the seroprevalence of T. gondii infection in horses and donkeys in Liaoning province, northeastern China. METHODS: Serum samples were collected from 711 and 738 slaughtered horses and donkeys, respectively, in 13 regions of Liaoning province. The modified agglutination test (MAT) was used to test the specific antibodies to T. gondii. RESULTS: In this study, 178 of 711 (25.0%) horses were seropositive for T. gondii with titers of 1:25 in 81, 1:50 in 62, 1:100 in 33, and 1:200 in 2, and seroprevalence of T. gondii infection from 13 regions ranged from 8.2% to 37.0%. Antibodies to T. gondii were found in 174 of 738 (23.6%) donkeys with titers of 1:25 in 66, 1:50 in 54, 1:100 in 49, and 1:200 in 5, and seroprevalence varied in 13 different regions, ranging from 9.1% to 32.6%. CONCLUSIONS: The results of the present study indicated that the rate of infection with T. gondii in horses and donkeys is a little high in Liaoning province, northeastern China in comparison to other surveys in China, which suggests that consumption of horse and donkey meat in this area may represent a potential source for human infection with T. gondii.

Seroprevalence of Toxoplasma gondii infection in slaughtered chickens, ducks, and geese in Shenyang, northeastern China.

BACKGROUND: In recent years, investigations of Toxoplasma gondii infection in poultry (chickens, ducks, and geese) have been reported worldwide, including China. However, little is known about the prevalence of T. gondii infection in poultry in northeastern China. Therefore, the present study was performed to determine the prevalence of T. gondii infection in slaughtered chickens, ducks, and geese in Shenyang, northeastern China. METHODS: In the present study, the seroprevalence of T. gondii in 502 adult chickens, 268 adult ducks, and 128 adult geese was surveyed using the modified agglutination test (MAT). RESULTS: The seroprevalences of T. gondii were 5.8%, 7.8%, and 4.7% in chickens, ducks, and geese, respectively. Prevalence was higher in free-range groups (11.2%, 12.3%, and 8.9%) than caged groups (4.7%, 7.5%, and 6.0%), and there was a statistically significant difference only between free-range chickens and caged chickens, but no significant difference was found between free-range ducks, geese and caged ducks, geese. CONCLUSIONS: The present study shows the prescence of T. gondii infection in slaughtered chickens, ducks, and geese in Shenyang, northeastern China, which suggests that consumption of poultry meat in Shenyang may pose a potential threat to human health and should be given attention.

[Inhibitory effects of suramin on growth of transplanted lung adencarcinoma in mice and its mechanisms].

BACKGROUND & OBJECTIVE: Recent study found that suramin could inhibit the growth of malignant tumors, but its in vivo effect on lung adenocarcinoma has seldom been reported. This study was to investigate the inhibitory effects of suramin on the growth and metastasis of transplanted lung adenocarcinoma in mice, and explore the mechanisms. METHODS: Lung adenocarcinoma LA795 cells were transplanted into 32 T739 mice. The tumor-bearing mice were randomized into control group, cisplatin (DDP) group, suramin group, and combination (suramin plus DDP) groupû each group contained 8 mice. Different treatments were served since Day 4 after transplantation. All mice were killed 24 days after transplantation. The metastatic tumor foci on the lung in mice were counted. The occurrence rate of lung metastasis and the inhibition rate of metastatic foci were calculated. The volume and weight of tumors were measured. The expression of epidermal growth factor receptor (EGFR), P-selectin and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected by immunhistochemistry. RESULTS: DDP and suramin used alone or in combination significantly inhibited the growth of LA795 cells in mice (P<0.05), with growth inhibition rates of 34.9%, 23.8% and 57.3%, respectively. The occurrence rate of lung metastasis and the number of metastatic foci on lung surface were significantly lower in suramin group and combination group than in DDP group and control group (P<0.05). The protein levels of EGFR and P-selectin were significantly higher in control group than in DDP group, suramin group, and combination group (157.7+/-6.1 vs. 130.7+/-5.9, 110.3+/-5.8, and 89.2+/-5.4, P<0.05û 134.5+/-5.7 vs. 117.9+/-5.1, 96.2+/-5.4, and 78.3+/-4.5, P<0.01). The S phase fraction of tumor cells was significantly higher in control group than in DDP group, suramin group, and combination group [(89.7+/-3.8)% vs. (68.8+/-4.0)%, (65.2+/-4.2)%, and (51.3+/-4.2)%, P<0.01)]. CONCLUSION: Suramin could inhibit the proliferation and metastasis of LA795 cells in T739 mice through regulating the expression of EGFR, P-selectin and PCNA.

[Effects of suramin in combination with cisplatin on growth and metastasis of lung adenocarcinoma xenografts in mice].

BACKGROUND & OBJECTIVE: Recent study found angiogenesis plays some important roles in tumor growth and metastasis. This study was to explore the inhibitory effect of angiogenesis inhibitor Suramin in combination with cisplatin (DDP) on the growth and lung metastasis of lung adenocarcinoma LA795 cell xenografts in mice. METHODS: Highly metastatic LA795 cells were inoculated into the mammary fatty pad of 32 T739 mice to establish lung adenocarcinoma models. Four days after inoculation, the mice were randomized into 4 groups: the mice in control group received intraperitoneal injection of 0.2 ml normal saline everyday; the mice in DDP group received injection of DDP [2 mg. (kg.day)-1] at the 4th, 11th, and 18th days; the mice in Suramin group received injection of Suramin [10 mg. (kg.day)-1] everyday; the mice in combination group received injection of DDP [2 mg. (kg.day)-1] at the 4th, 11th, and 18th days, and Suramin [10 mg. (kg day)-1] everyday. All mice were killed 24 days later. Lung and subcutaneous tumors were examined histologically. The metastatic tumor foci on lung surface were observed, lung weight was measured, the occurrence rate of lung metastasis and the inhibitory rate of metastatic foci were calculated, subcutaneous tumor microvessel density (MVD), vascular endothelial growth factor (VEGF), and nuclear factor-kappaB (NF-kappaB) were determined by immunohistochemistry, and tumor cell apoptosis was measured by TUNEL method. RESULTS: In DDP, Suramin, and combination groups, tumor growth was suppressed significantly, with growth inhibitory rates of 23.0%, 34.4%, and 56.3%, respectively (P<0.05). Necrosis and decrease of tumor vessels were observed in Suramin and combination groups. The expression of NF-kappaB was significantly lower and tumor cell apoptosis index was significantly higher in DDP, Suramin, and combination groups than in control group (P<0.01). The metastatic foci on lung surface were less in Suramin and combination groups than in DDP and control groups. The expression of MVD and VEGF in subcutaneous tumors and the occurrence rate of lung metastasis were also obviously lower in Suramin and combination groups. CONCLUSION: Suramin has synergetic inhibitory effect with DDP on growth and metastasis of lung adenocarcinoma LA795 cell xenografts in mice through inhibiting angiogenesis and inducing cell apoptosis.

[Inhibitory effect of fumagillol combined with cyclophosphamide on metastasis of lung adenocarcinoma cell line LA795 xenograft in mice].

BACKGROUND & OBJECTIVE: Treating tumor with angiogenesis inhibitor and chemotherapeutic drugs is a research hot spot now. This study was designed to observe the synergetic inhibitory effect of fumagillol (TNP-470) in combination with cyclophosphamide (CTX) on metastasis of lung adenocarcinoma cell line LA795 xenograft in mouse, and to explore the related mechanism of suppressing tumor metastasis by TNP-470. METHODS: Forty T739 nude mice bearing highly metastatic LA795 cells were randomized into 5 groups: control group, vehicle group, TNP-470 (30 mg/kg) group, CTX (40 mg/kg) group, and combination group (TNP-470 plus CTX). All mice were killed 3 weeks laterû the subcutaneous tumors were weighted to calculate inhibitory rate. The metastatic tumor foci on lung surface in mice were counted to calculate occurrence rate and inhibitory rate of metastases on lung surface. The microvessel density (MVD) and the expression of tumor metastasis-related factor P-selectin in subcutaneous tumor were detected by immunohistochemistry and analyzed with image analysis system. RESULTS: The inhibitory rate of tumor was significantly higher in combination group (81.5%) than in other groups (P<0.01). TNP-470 plus CTX showed synergetic effect on inhibiting metastasis on lung surface with a Q value of 1.21. The metastatic foci on lung surface were significantly fewer in combination group, TNP-470 group, and CTX group than in control group (1.75+/-1.71, 4.75+/-3.34, and 8.50+/-2.67 vs. 12.13+/-4.02, P<0.05). The MVD and the expression of P-selectin in subcutaneous tumor were also significantly lower in combination group and TNP-470 group than in control group (9.13+/-1.61 and 12.13+/-2.84 vs. 20.50+/-3.12, P<0.01; 5.25+/-2.27 and 7.13+/-3.01 vs. 13.75+/-3.38, P<0.01). CONCLUSIONS: TNP-470 and CTX have synergetic inhibitory effect on lung metastasis of LA795 xenograft tumor. TNP-470 may inhibit lung metastasis of LA795 xenograft tumor by suppressing the expression of P-selectin.