Protective effects of crocin against gentamicin-induced damage in rat testicular tissue: Modulating the levels of NF-κB/TLR-4 and Bax/Bcl-2/caspase-3 signaling pathways.

Objective To investigate the expression and significance of Toll-like receptor 4 (TLR4) in renal tissue and peripheral blood of children with idiopathic nephrotic syndrome(INS). Methods The renal biopsy tissues of 78 children with INS diagnosed in the First Affiliated Hospital of Xinxiang Medical University from October 2015 to June 2018 and normal renal tissues of 21 children (control group 1) were collected, and the expressions of TLR4 in the renal tissue was detected by using immunohistochemical method.The expression of TLR4 in different renal pathological types and clinical types of INS was compared, and the correlation of TLR4 with 24-hour urinary protein and serum albumin was analyzed.The expression levels of TLR4 in peripheral blood of children with INS before and after treatment (active stage and remission stage) and 23 healthy children (control group 2) were detected by enzyme linked immunosorbent assay(ELISA). The serum expression levels of TLR4 in different renal pathological types and clinical types of INS were compared, and the correlation of TLR4 with 24-hour urinary protein and serum albumin was analyzed; The correlation between TLR4 expression in renal tubules and in the serum of children with INS was also analyzed. Results (1)Compared with the expression of TLR4 in normal renal tissues[(0.93±0.26)%], the expression of TLR4 in glomeruli and interstitium of all pathological types of INS [mesangial proliferative glomerulonephritis (MsPGN): (0.93 ± 0.21)%, focal segmental glomerulosclerosis (FSGS): (1.02±0.25)%, membranous glomerulonephritis(MN): (1.03±0.09)%, minimal change disease(MCD): (1.02±0.27)%]was not significantly different (F=0.741, P=0.562), but the expression of TLR4 in renal tubules[MCD: (82.94±4.62)%, MN: (63.54±1.98)%, MsPGN(42.32±2.97)%, FSGS: (22.60±2.07)%] was significantly increased (F=1 929.842, P<0.01), Especially, the expression of TLR4 in renal tubules of MCD type INS was significantly higher than that of MN, MsPG N and FSGS [MCD: (82.94±4.62)%, MN: (63.54±1.98)%, MsPGN: (42.32±2.97)%, FSGS: (22.60±2.07)%], and the differences were statistically significant(all P<0.01). TLR4 expression in renal tubules was the highest in steroid-sensitive nephrotic syndrome (SSNS) type and the lowest in INS patients with steroid-resistant nephrotic syndrome (SRNS) type, and the differences were statistically significant(F=220.951, P<0.01). (2)The expression of serum TLR4 in INS children at the active stage [MsPNG: (143.36±12.99) ng/L, FSGS(75.94±7.29) ng/L, MN(210.22±14.66) ng/L, MCD(283.93±21.58) ng/L]was significantly higher than that in INS children at remission stage [MsPNG: (29.51±4.93) ng/L, FSGS(15.66±3.78) ng/L, MN(45.40±5.73) ng/L, MCD(62.29±7.90) ng/L]and control group 2[(0.69 ± 0.33) ng/L], and the differences were statistically significant(all P<0.01); the expression of serum TLR4 in INS children at remission stage was significantly higher than that in the control group 2 (F=286.287, P<0.01). TLR4 had the highest expression level in serum of MCD type INS children at active and remission stages, followed by MN and FSGS successively.The expression of serum TLR4 was highest in SSNS and lowest in SRNS, and the differences were statistically significant (F=147.438, P<0.01). (3)The expression of TLR4 in renal tubules of children with INS[(62.82 ±20.94)%]was positively correlated with the expression of TLR4 in serum[(213.26±73.33) ng/L] (r=0.852, P< 0.05). The expression levels of TLR4 in renal tubules and serum of INS patients at active stage were positively correlated with 24-hour urinary protein level[(123.05±33.55) mg/kg] (r=0.401, 0.427, all P<0.05), and negatively correlated with serum albumin level[(19.54±3.55)g/L] (r=-0.602, -0.617, all P<0.05). Conclusions The expression of TLR4 in renal tubules and serum of children with INS increases, and may be related to different renal pathological types and clinical types of children with INS, as well as disease activity. Key words: Toll-like receptor 4; Kidney tissue; Serum; Idiopathic nephrotic syndrome; Child

The potential ameliorative effect of curcumin nanoemulsion on busulfan-induced testicular toxicity in adult rat model.

Aroclor 1260 is one of the more representative polychlorinated biphenyls found in biota. This study was designed to delineate the testicular toxicity of Aroclor 1260 and to elucidate the potential protective role of Calligonum comosum (C. comosum) and lipoic acid in adult rats. Aroclor 1260 was dissolved in corn oil and given to rats by gavage at doses 0, 20, 40, or 60 mg/kg/day for 15 consecutive days (Groups I, II, III, and IV, respectively). Groups V and VI were pretreated with C. comosum (200 mg/kg/day) and lipoic acid (35 mg/kg/day) respectively 24 h before Aroclor 1260 (40 mg/kg/day) treatment for 15 consecutive days. Aroclor 1260 (20, 40 or 60 mg/kg/day) treatment significantly decreased testes weight, sperm count and motility and daily sperm production. Serum testosterone was significantly decreased in response to treatment with 40 and 60 mg/kg/day of Aroclor 1260. LDH-X activity was significantly decreased at the three dose levels. Hydrogen peroxide (H2 O2 ) production (in a dose-related manner) and lipid peroxidation were significantly increased in response to Aroclor 1260 (20, 40, or 60 mg/kg/day) treatment. Aroclor 1260 at the three dose levels decreased the activities of the antioxidant enzymes SOD, CAT, GPx, and GR and the non-enzymatic antioxidant GSH level. CAT, GPx and GSH showed a dose-response effect. These abnormalities were effectively attenuated by pretreatment with C. comosum (200 mg/kg/day) or lipoic acid (35 mg/kg/day). Histopathological examination showed a dose-related increase in morphological abnormalities of the testis in response to Aroclor 1260 treatment. In conclusion, Aroclor 1260 induced testicular toxicity at least, in part, by induction of oxidative stress. By reversal of biochemical and morphological changes towards normalcy, the cytoprotective role of C. comosum and lipoic acid is illuminated. In comparison, lipoic acid was more protective than C. comosum extract against testicular toxicity induced by Aroclor 1260. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1147-1157, 2017.

CD73 is a 5' ectonuclease that catalyzes the conversion of cyclic AMP into the highly immunosuppressive adenosine in extracellular space. In addition to the cells of the immune system, CD73 is highly expressed in various cancer cell lines and clinical cancer tissues. Toll like receptor-9 (TLR9) is a cellular DNA-receptor that is highly expressed in breast cancer. Both CD73 and TLR9 expression have recently been associated with TNBC prognosis but the mechanisms how these proteins possibly contribute to TNBC pathophysiology remains poorly understood. TLR9 and CD73 expression has been shown to be mutually regulated in various cell types. Whether this is the case in cancer is unknown. The aim of this study was to investigate the mutual role of TLR9 and CD73 in breast cancer (BC). Specifically our hypothesis was that TLR9 and CD73 expression correlate in TNBC. We compared immunohistological tumor TLR9 and CD73 expression scores using a previously characterized breast cancer (BC) cohort (n=184) with follow-up time of &amp;gt; 10 years. We did not discover a connection between TLR9 and CD73 expression in tumor cells in BC. There was a trend for increased survival among patients that had high tumor cell CD73 expression, as compared with the lower tumor cell CD73 expression groups. There was a trend for a better survival among TNBC patients that had lower stromal CD73 expression, as compared with those TNBC patients that had higher stromal CD73 expression. No such difference was detected among patients with non-TNBC tumors. Our results suggest that stromal vs. tumor cell CD73 expression have opposite effects on survival in TNBC, but there is no connection between CD73 and TLR9 expression. Our conclusions are limited by low sample numbers. Citation Format: Selander K, Mella M, Kauppila J, Karihtala P, Jukkola-Vuorinen A, Auvinen P, Soini Y, Kauppila S, Haapasaari K-M, Harris K, Vuopala K. Comparison of tumor and stroma CD73 expression with TLR9 and survival in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-13.

Objective To evaluate the role of caveolin-1 (Cav-1) in penehyclidine hydrochioride(PHC)-induced inhibition of lipopolysaccharide(LPS)-induced activation of Toll-like receptor 4 (TLR4)/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in macrophages of mice. Methods Macrophages of mice were seeded in 6 cm diameter dishes (5 ml per dish) and divided into 5 groups (n=20 each) using a random number table: Scr-siRNA group (S group), Scr-siRNA+ LPS group (LPS group), Scr-siRNA+ LPS + PHC group (LPS+ P group), Cav-1-siRNA+ LPS group (C+ LPS group) and Cav-1-siRNA+ LPS+ PHC group (C+ LPS+ P group). Macrophages were transfected with Scr-siRNA for 24 h in S, LPS and LPS+ P groups and with Smart pool Cav-1 siRNAs for 24 h in C+ LPS and C+ LPS+ P groups.LPS at the final concentration of 1 μg/ml was added after the end of transfection, and macrophages were then incubated for 2 h in LPS, LPS+ P, C+ LPS and C+ LPS+ P groups.In LPS+ P and C+ LPS+ P groups, PHC at the final concentration of 2 μg/ml was added at 2 h of incubation with LPS, and macrophages were then incubated for 2 h. The expression of Cav-1 and TLR4 was detected by Western blot.The expression of p38 MAPK was determined by immunofluorescence.The level of tumor necrosis factor-alpha (TNF-α) in the culture medium was determined by enzyme-linked immunosorbent assay.The activity of myeloperoxidase (MPO) in macrophages was measured by colorimetry. Results Compared with group S, the expression of TLR4 and p38 MAPK was significantly up-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were increased in the other four groups, the expression of Cav-1 was significantly down-regulated in LPS and C+ LPS groups (P 0.05). Compared with group LPS, the expression of Cav-1 was significantly up-regulated, the expression of TLR4 and p38 MAPK was down-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were decreased in group LPS+ P, and the expression of Cav-1 was significantly down-regulated, the expression of TLR4 and p38 MAPK was up-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were increased in group C+ LPS (P<0.05). Compared with group LPS+ P, the expression of Cav-1 was significantly down-regulated, the expression of TLR4 and p38 MAPK was up-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were increased in group C+ LPS+ P (P<0.05). Compared with group C+ LPS, the expression of Cav-1 was significantly up-regulated, the expression of TLR4 and p38 MAPK was down-regulated, and the concentration of TNF-α in the culture medium and activity of MPO were decreased in group C+ LPS+ P (P<0.05). Conclusion The mechanism by which PHC inhibits LPS-induced activation of TLR4/p38 MAPK signaling pathway in macrophages is related to up-regulating Cav-1 expression in mice. Key words: Caveolin 1; Cholinergic antagonists; Lipopolysaccharides; Toll-like receptor 4; p38 Mitogen-activated protein kinases; Macrophages

Monocyte toll-like receptor 4 (TLR4) has been implicated in the pathogenesis of atherosclerosis with increased levels in myocardial infarction. The aim of this study was to assess the numbers of TLR4(+) monocytes in each monocyte subset in MI, the expression of TLR4 and association with markers of monocyte activation, inflammation, myocardial damage and postmyocardial infarction (MI) cardiac contractility. Surface expression of TLR4 and numbers of TLR4-expressing monocytes were quantified by flow cytometry of venous blood in 50 patients with ST-elevation MI (STEMI), 48 with non-STEMI (NSTEMI) and 40 with stable coronary artery disease (CAD). These parameters were measured on days 1, 3, 7 and 30 post-MI in STEMI patients. Three monocyte subsets were defined as CD14(++) CD16(-) CCR2(+) (Mon1), CD14(++) CD16(+) CCR2(+) (Mon2) and CD14(+) CD16(++) CCR2(-) (Mon3). Plasma inflammatory cytokines were assessed using cytometric bead arrays. There was a significant increase in counts of TLR4(+) Mon1 and Mon2 in STEMI patients and TLR4(+) Mon2 in NSTEMI patients compared with controls with CAD. Monocyte TLR4(+) expression was similar between the groups, and was not changed during follow-up in STEMI patients. Plasma interleukin-6 (IL6) levels correlated positively with TLR4(+) Mon2 count (r=0.54, P<0.001), but negatively with TLR4 expression on Mon2 (r=-0.33, P=0.021). Following treatment of acute MI, TLR4 expression by individual monocyte subsets is unchanged. An increase in TLR4(+) Mon1 and Mon2 count in patients with STEMI and TLR(+) Mon2 count in those with NSTEMI is due to an increase in monocyte subset count and not to changes in TLR4 expression. Monocyte counts but not TLR4 expression correlate positively with plasma IL6 levels. We suggest that TLR4 expression may not be a reliable marker of monocyte activation in MI.

Objective To evaluate the effect of dexmedetornidine on the expression of Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) in the spinal cord in rats with neuropathic pain (NP).Methods One hundred and eight male Wistar rats,aged 6-8 weeks,weighing 180-220 g,were randomly assigned into 3 groups (n =36 each):sham operation group (group S),NP group and dexmedetomidine group (group D).NP was induced by chronic constrictive injury in anesthetized rats.Sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread.In group S,the right sciatic nerves were exposed,but not ligated.Dexmedetomidine 50 μg/kg was injected intraperitoneally once a day from the onset of operation to one day before the rats were sacrificed in group D,while the equal volume of normal saline was injected in groups S and NP.Mechanical withdrawal threshold (MWT) and thermal pain threshold (TPT) were measured on the day before operation (T0) and 3,7,and 14 days after operation (T1-3).After measurement of pain threshold at T1,T2 and T3 after operation,the L4-6 segments of the spinal cord were removed for determination of the expres-sion of TLR4 and NF-κB mRNA (by RT-PCR) and the expression of TLR4 and NF-κB in spinal dorsal horn (by immuno-histochemistry).Results Compared with group S,MWT and TPT were significantly decreased and the expression of TLR4,NF-κB and TLR4 and NF-κB mRNA was up-regulated after operation in groups NP and D (P ＜ 0.05).Compared with group NP,TPT and MWT were significantly increased and the expression of TLR4,NF-κB,TLR4 mRNA and NF-κB mRNA was significantly down-regulated after operation in group D (P ＜ 0.05).Conclusion The mechanism by which dexmedetomidine attenuates NP in rats is related to inhibition of the expression of TLR4 and NF-κB in rat spinal cord. Key words: Dexmedetomidine ; Neuralgia ; Toll-like receptor 4 ; NF-kappa B

Increased expression of Toll-like receptors 3, 7, 8 and 9 in peripheral blood mononuclear cells in patients with psoriasis.

W1816 Loss of TLR 2 and 4 on Ileal Plasmacytoid Dendritic Cells in Post-Operative Crohn's Disease Patients

The aim of the present study was to investigate the potential oxidative damage of di-(2-ethylhexyl) phthalate (DEHP) in the rat testis and to further elucidate the potential modulatory effect of quercetin. DEHP was diluted in corn oil and given to rats by oral gavage at doses 0, 300, 600, and 900 mg/kg/day (groups I, III, IV, or V, respectively) for 15 consecutive days. Group VI was pretreated with quercetin (90 mg/kg), 24 h before starting the experiment and then treated with DEHP (900 mg/kg/day) for 15 consecutive days. Group II was treated with quercetin (90 mg/kg/day). The relative testes weight and sperm motility were significantly decreased by treatment with 900 mg/kg of DEHP. Both sperm count and daily sperm production were significantly decreased by DEHP treatment at doses of 600 and 900 mg/kg. Serum testosterone level and prostatic acid phosphatase (ACP) activity and testicular lactate dehydrogenase-X (LDH-X) activity were significantly decreased in animals treated with 900 mg/kg. Serum total ACP activity was significantly increased in animals treated with 600 and 900 mg/kg of DEHP. DEHP treatment induced oxidative stress and histopathological abnormality. These abnormalities were effectively normalized by pretreatment with quercetin except for LDH-X near normalcy. In conclusion, the findings of this study demonstrate that DEHP impairs testicular function at least, in part, by inducing oxidative stress and quercetin has a potent protective effect against DEHP-induced testicular toxicity in rats.

Arsenic, acting as an endocrine disruptor, causes reproductive malfunctions. Studies have been undertaken to find out whether the co-supplementation of α-tocopherol and ascorbic acid (AT-AA) could reduce the arsenic-induced testicular toxicity caused by oxidative stress and resulting DNA damage. Adult male Wistar rats (120±10 g) were given arsenic trioxide [3 mg/kg body weight (b.wt.) per day] for 30 consecutive days and the supplement group received α-tocopherol (400 mg/kg b.wt. per day) and ascorbic acid (200 mg/kg b.wt. per day). Reproductive functions were evaluated with respect to the histoarchitecture, gametokinetic activity, androgenic potential, glutathione-dependent antioxidant status and DNA damage of the testis. Arsenic treatment caused marked reduction in the relative weight of the testis (p<0.05) but showed no effect on body weight. The number of germ cells at stage VII of the spermatogenic cycle (p<0.01), the seminiferous tubular diameter (p<0.001) and Leydig cell nuclear area (p<0.01) were significantly reduced. Notable decrease in the activities of testicular Δ5, 3β-HSD (p<0.05) and 17β-HSD (p<0.01) with a concomitant fall in serum testosterone level (p<0.01) along with significant diminution in testicular glutathione S-transferase (p<0.05) activity and reduced glutathione level (p<0.01) were observed. Significant DNA damage (p<0.001) in spermatogenic cells was also noted. All these alterations including DNA strand breakage were seen to be protected with the coadministration of AT-AA. The data suggest that the protection of testicular toxicity in arsenic-exposed adult rats is possible with combined coadministration of AT-AA.

Several organ systems can be affected by nicotine/cigarette smoking (CS); however, there is a gap of knowledge about the role of local neurotransmitter system, brain-derived neurotrophic factor (BDNF), and dopamine (DA) in testicular toxicity. Therefore, the aim of the present study is to explore the toxic impact of short- and long-term exposure to oral nicotine and passive CS on adult albino Wistar rats, using doses that closely mimic the human smoking scenario. Our results showed dose- and time-dependent loss of developing spermatogonia and spermatocyte of the seminiferous tubules, disruption of basement membrane, DNA damage, and high serum cotinine upon exposure to nicotine and CS, resulting in low sperm count as compared to control. Further, the results showed the upregulation of BDNF, DA, tyrosine hydroxylase (TH), and pro-oxidants, ie, reactive oxygen species (ROS) and inducible nitric oxide synthetase (iNOS), in the exposed testis and downregulation of the antioxidants such as, ascorbate and Nrf2 when compared with the control. Thus, our results for the first time highlight a potential role of the local neurotransmitter system and antioxidant depletion (Nrf2) in nicotine/CS-induced testicular pathogenesis, which could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders, and probably establish a link with the brain system contributing to addiction.

Objective To evaluate the effects of penehyclidine hydrochloride (PHC) on the expression of Toll like receptor-4 (TLR4) and nuclear factor kappa B (NF-κcB) in a rat model of sepsis-induced acute lung injury (ALI).Methods Seventy-two adult male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 3 groups (n =24 each) using a random number table:shame operation group (group S),ALI group and PHC group.Sepsis-induced ALl was induced by cecal ligation and puncture in anesthetized rats.In group PHC,PHC 0.45 mg/kg was intramuscularly injected immediately after cecal ligation and puncture.At 6,12,24 and 36 h after ligation,the broncho-alveolar lavage fluid (BALF) was collected for detection of TNF-α and IL-6 concentrations and the lungs were removed for determination of the expression of TLR4 (using RT-PCR and Western blot) and NF-κBp65 (using Western blot) in lung tissues and for microscopic examination.The pathological changes of lungs were scored.The wet to dry lung weight (W/D) ratio was calculated and the activity of myeloperoxidase (MPO) in lung tissues was measured.Results As compared with S group,the IL-6 concentrations in BALF at 6,12 and 24 h after ligation,TNF-α concentration in BALF at 6 and 12 h after ligation,and the expression of TLR4 and NF-κBp65,pathological scores,W/D ratio and MPO activity at each time point were significantly increased in ALI group (P ＜ 0.05).Compared with ALI group,the TNF-α concentration in BALF at 6 and 12 h after ligation,and IL-6 concentrations in BALF,the expression of TLR4 and NF-κBp65,pathological scores,W/D ratio and MPO activity at each time point were significantly decreased in group PHC (P ＜ 0.05).Conclusion PHC inhibits NF-κB activation and decreases inflammatory responses through blocking TLR4 expression in lung tissues,thus attenuating sepsis-induced ALI in rats. Key words: Cholinergic antagonists; Sepsis; Respiratory distress syndrome, adult ; Toll-like receptor 4; NF-kappa B

Funding Acknowledgements Type of funding sources: None. Background Atherosclerosis is inherently an inflammatory process, with a complex interplay of inflammatory markers. It is established that these inflammatory markers play an important role in patients of Coronary Artery Disease(CAD) with traditional cardiovascular risk factors. However, the role of inflammation in the atherosclerotic process in patients of CAD without traditional risk factors is still not clearly known. Purpose Our purpose was to determine whether in patients of CAD without traditional risk factors, TLR4(Toll like receptor 4) expression as a marker of inflammation is similar to that in patients of CAD with traditional risk factors. Materials and Methods This observational cross sectional study was done between July’20 to Dec’21. Equal number of patients of CAD with and without traditional cardiovascular risk factors undergoing Coronary Artery Bypass Grafting (CABG) were enrolled. The risk factors considered were Hypertension, Diabetes, Dyslipidemia, Obesity and Addiction to Tobacco and/or Alcohol. A minimum of two punch biopsy samples of aortic tissue was taken from each subject undergoing CABG. Immunohistochemistry for TLR4, was done in Ventana BenchMark GX System. The primary TLR4 antibody was procured from reputed source. Results The presence or absence of TLR4 expression was associated significantly with the Syntax scores (37.40±4.74 vs 29.5±8.71; p value=0.036), total Cholesterol (187±35.06mg/dL vs 130±35.69 mg/dL ;p value 0.010) and LDL cholesterol (118.86±28.12mg/dL vs 64.21±25.61 mg/dL; p value 0.003). TLR4 expression, however, was not significantly associated with the number of coronary vessels involved (p=0.298). TLR4 expression was also not significantly associated with any other individual risk factors. However, when the traditional risk factors were considered in combination, TLR4 expression was associated significantly with the number of risk factors present(p=0.029) the strongest being in those having 4 traditional risk factors. The level of TLR4 expression gradually declined with the decrease in the number of risk factors, having mostly weak or negative expression in patients without any traditional risk factors. Conclusion Patients with CAD without any traditional risk factors, had a less severe coronary artery disease as manifested by lower Syntax scores, and had lower degree of TLR4 expression. Patients with CAD with traditional risk factors had more severe coronary artery disease as evidenced by higher Syntax scores, and had higher degree of TLR4 expression proportional to the number of traditional risk factors present. Thus, differential TLR4 expression in CAD patients with and without traditional risk factors indicated a difference in inflammatory state between the two groups and warrants further investigation.

Inhibiting expression of HSP60 and TLR4 attenuates paraquat-induced microglial inflammation