Procyanidin B2 improves developmental capacity of bovine oocytes via promoting PPARγ/UCP1-mediated uncoupling lipid catabolism during in vitro maturation.

Metabolic balance is essential for oocyte maturation and acquisition of developmental capacity. Suboptimal conditions of in vitro cultures would lead to lipid accumulation and finally result in disrupted oocyte metabolism. However, the effect and mechanism underlying lipid catabolism in oocyte development remain elusive currently. In the present study, we observed enhanced developmental capacity in Procyanidin B2 (PCB2) treated oocytes during in vitro maturation. Meanwhile, reduced oxidative stress and declined apoptosis were found in oocytes after PCB2 treatment. Further studies confirmed that oocytes treated with PCB2 preferred to lipids catabolism, leading to a notable decrease in lipid accumulation. Subsequent analyses revealed that mitochondrial uncoupling was involved in lipid catabolism, and suppression of uncoupling protein 1 (UCP1) would abrogate the elevated lipid consumption mediated by PCB2. Notably, we identified peroxisome proliferator-activated receptor gamma (PPARγ) as a potential target of PCB2 by docking analysis. Subsequent mechanistic studies revealed that PCB2 improved oocyte development capacity and attenuated oxidative stress by activating PPARγ mediated mitochondrial uncoupling. Our findings identify that PCB2 intricately improves oocyte development capacity through targeted activation of the PPARγ/UCP1 pathway, fostering uncoupling lipid catabolism while concurrently mitigating oxidative stress.

Design, synthesis, and bioevaluation of SOS1 PROTACs derived from pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor.

Oncogenic KRAS mutations drive an approximately 25 % of all human cancers. Son of Sevenless 1 (SOS1), a critical guanine nucleotide exchange factor, catalyzes the activation of KRAS. Targeting SOS1 degradation has engaged as a promising therapeutic strategy for KRAS-mutant cancers. Herein, we designed and synthesized a series of novel CRBN-recruiting SOS1 PROTACs using the pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor as the warhead. One representative compound 11o effectively induced the degradation of SOS1 in three different KRAS-mutant cancer cell lines with DC50 values ranging from 1.85 to 7.53 nM. Mechanism studies demonstrated that 11o-induced SOS1 degradation was dependent on CRBN and proteasome. Moreover, 11o inhibited the phosphorylation of ERK and displayed potent anti-proliferative activities against SW620, A549 and DLD-1 cells. Further optimization of 11o may provide us promising SOS1 degraders with favorable drug-like properties for developing new chemotherapies targeting KRAS-driven cancers.

Comparison of Umbilical Cord Mesenchymal Stem Cells and Fibroblasts as Donor Nuclei for Handmade Cloning in Sheep Using a Single-Cell Transcriptome.

Oocytes are efficient at reprogramming terminally differentiated cells to a totipotent state. Nuclear transfer techniques can exploit this property to produce cloned animals. However, the overall efficiency is low. The use of umbilical cord mesenchymal stem cells (UC-MSCs) as donor nuclei may increase blastocyst rates, but the exact reasons for this remain unexplored. A single-cell transcriptomic approach was used to map the transcriptome profiles of eight-cell embryos that were in vitro-fertilized and handmade-cloned using umbilical cord mesenchymal stem cells and fibroblasts as nuclear donors. Differences were examined at the chromatin level, the level of differentially expressed genes, the level of histone modifications and the level of DNA methylation. This research provides critical information regarding the use of UC-MSCs as a preferred donor nucleus for nuclear transfer techniques. It also offers unique insights into the mechanism of cellular reprogramming.

Design, Synthesis, and Biological Evaluation of Potent and Selective PROTAC Degraders of Oncogenic KRASG12D.

KRASG12D, the most frequent KRAS oncogenic mutation, is a promising target for cancer therapy. Herein, we report the design, synthesis, and biological evaluation of a series of KRASG12D PROTACs by connecting the analogues of MRTX1133 and the VHL ligand. Structural modifications of the linker moiety and KRAS inhibitor part suggested a critical role of membrane permeability in the degradation activity of the KRASG12D PROTACs. Mechanism studies with the representative compound 8o demonstrated that the potent, rapid, and selective degradation of KRASG12D induced by 8o was via a VHL- and proteasome-dependent manner. This compound selectively and potently suppressed the growth of multiple KRASG12D mutant cancer cells, displayed favorable pharmacokinetic and pharmacodynamic properties in mice, and showed significant antitumor efficacy in the AsPC-1 xenograft mouse model. Further optimization of 8o appears to be promising for the development of a new chemotherapy for KRASG12D-driven cancers as the complementary therapeutic strategy to KRAS inhibition.

Vitrification of Mammalian Oocytes: Recent Studies on Mitochondrial Dysfunction.

Vitrification of reproductive cells is definitely essential and integral in animal breeding, as well as in assisted reproduction. However, issues accompanied with this technology such as decreased oocyte competency and relatively low embryo survival rates appear to be a tough conundrum that has long perplexed us. As significant organelles in cell metabolism, mitochondria play pivotal roles in numerous pathways. Nonetheless, extensive evidence has demonstrated that vitrification can seriously impair mitochondrial function in mammalian oocytes. Thus, in this article, we summarize the current progress in oocyte vitrification and particularly outline the common mitochondrial abnormalities alongside subsequent injury cascades seen in mammalian oocytes following vitrification. Based on existing literature, we tentatively come up with the potential mechanisms related to mitochondrial dysfunction and generalize efficacious ways which have been recommended to restore mitochondrial function.

Integrated transcriptomics and proteomics assay identifies the role of FCGR1A in maintaining sperm fertilization capacity during semen cryopreservation in sheep.

Semen cryopreservation is a promising technology employed in preserving high-quality varieties in animal husbandry and is also widely applied in the human sperm bank. However, the compromised qualities, such as decreased sperm motility, damaged membrane structure, and reduced fertilization competency, have significantly hampered the efficient application of this technique. Therefore, it is imperative to depict various molecular changes found in cryopreserved sperm and identify the regulatory network in response to the cryopreservation stress. In this study, semen was collected from three Chinese Merino rams and divided into untreated (fresh semen, FS) and programmed freezing (programmed freezing semen, PS) groups. After measuring different quality parameters, the ultra-low RNA-seq and tandem mass tag-based (TMT) proteome were conducted in both the groups. The results indicated that the motility (82.63% ± 3.55% vs. 34.10% ± 2.90%, p < 0.05) and viability (89.46% ± 2.53% vs. 44.78% ± 2.29%, p < 0.05) of the sperm in the FS group were significantly higher compared to those in the PS group. In addition, 45 upregulated and 291 downregulated genes, as well as 30 upregulated and 48 downregulated proteins, were found in transcriptomics and proteomics data separately. Moreover, three integrated methods, namely, functional annotation and enrichment analysis, Pearson's correlation analysis, and two-way orthogonal partial least squares (O2PLS) analysis, were used for further analysis. The results suggested that various differentially expressed genes and proteins (DEGs and DEPs) were mainly enriched in leishmaniasis and hematopoietic cell lineage, and Fc gamma receptor Ia (FCGR1A) was significantly downregulated in cryopreserved sperm both at mRNA and protein levels in comparison with the fresh counterpart. In addition, top five genes (FCGR1A, HCK, SLX4, ITGA3, and BET1) and 22 proteins could form a distinct network in which genes and proteins were significantly correlated (p < 0.05). Interestingly, FCGR1A also appeared in the top 25 correlation list based on O2PLS analysis. Hence, FCGR1A was selected as the most potential differentially expressed candidate for screening by the three integrated multi-omics analysis methods. In addition, Pearson's correlation analysis indicated that the expression level of FCGR1A was positively correlated with sperm motility and viability. A subsequent experiment was conducted to identify the biological role of FCGR1A in sperm function. The results showed that both the sperm viability (fresh group: 87.65% ± 4.17% vs. 75.8% ± 1.15%, cryopreserved group: 48.15% ± 0.63% vs. 42.45% ± 2.61%, p < 0.05) and motility (fresh group: 83.27% ± 4.15% vs. 70.41% ± 1.07%, cryopreserved group: 45.31% ± 3.28% vs. 35.13% ± 2.82%, p < 0.05) were significantly reduced in fresh and frozen sperm when FCGR1A was blocked. Moreover, the cleavage rate of embryos fertilized by FCGR1A-blocked sperm was noted to be significantly lower in both fresh (95.28% ± 1.16% vs. 90.44% ± 1.56%, p < 0.05) and frozen groups (89.8% ± 1.50% vs. 82.53% ± 1.53%, p < 0.05). In conclusion, our results revealed that the downregulated membrane protein FCGR1A can potentially contribute to the reduced sperm fertility competency in the cryopreserved sheep sperm.

Discovery of a Potent, Cooperative, and Selective SOS1 PROTAC ZZ151 with In Vivo Antitumor Efficacy in KRAS-Mutant Cancers.

The linker moiety of a proteolysis-targeting chimera (PROTAC) molecule plays a critical role in modulating the degradation activity, target selectivity, and physico-chemical properties. However, the basics and underlying mechanisms of chemical modifications of the linker structure causing dramatic changes in the PROTAC degradation activity warrant further investigation. Herein, we report the design and characterization of a highly potent and selective SOS1 PROTAC ZZ151. After systematically modifying the linker length and composition, we observed that subtle modification of just one atom of the linker moiety of ZZ151 resulted in remarkable changes in the formation of the ternary complex and thus dramatically affected the degradation activities. ZZ151 quickly, specifically, and effectively induced SOS1 degradation; displayed potent antiproliferation activities against a broad panel of KRAS mutant-driven cancer cells; and showed superior anticancer activities in the KRASG12D- and G12V-mutant xenografts in mice. ZZ151 is a promising lead for developing new chemotherapies targeting KRAS mutants.

Design, Synthesis, and Biological Evaluation of Bipyridazine Derivatives as Stimulator of Interferon Genes (STING) Receptor Agonists.

The development of stimulator of interferon genes (STING) agonists has been of potential applications for the treatment of cancer and infectious diseases. Based on the crystal structure of SR-717 bound to hSTING, we designed and synthesized a novel series of bipyridazine derivatives as highly potent STING agonists. Among them, compound 12L led to significant thermal stability shifts of the common alleles of hSTING, as well as that of mSTING. 12L also displayed potent activities in various hSTING alleles and mSTING competition binding assay. Specifically, 12L displayed higher cell-based activities than SR-717 in both human THP1 (EC50 = 0.38 ± 0.03 µM) and mouse RAW 264.7 cells (EC50 = 12.94 ± 1.78 µM), and was validated to activate the downstream signaling pathway of STING via a STING-dependent manner. Furthermore, compound 12L showed favorable pharmacokinetic (PK) properties and antitumor efficacy. These findings suggested that compound 12L has development potential as an antitumor agent.

Design, Synthesis, and Bioevaluation of Pyrido[2,3-d]pyrimidin-7-ones as Potent SOS1 Inhibitors.

The use of small molecular modulators to target the guanine nucleotide exchange factor SOS1 has been demonstrated to be a promising strategy for the treatment of various KRAS-driven cancers. In the present study, we designed and synthesized a series of new SOS1 inhibitors with the pyrido[2,3-d]pyrimidin-7-one scaffold. One representative compound 8u showed comparable activities to the reported SOS1 inhibitor BI-3406 in both the biochemical assay and the 3-D cell growth inhibition assay. Compound 8u obtained good cellular activities against a panel of KRAS G12-mutated cancer cell lines and inhibited downstream ERK and AKT activation in MIA PaCa-2 and AsPC-1 cells. In addition, it displayed synergistic antiproliferative effects when used in combination with KRAS G12C or G12D inhibitors. Further modifications of the new compounds may give us a promising SOS1 inhibitor with favorable druglike properties for use in the treatment of KRAS-mutated patients.

Leonurine improves bovine oocyte maturation and subsequent embryonic development by reducing oxidative stress and improving mitochondrial function.

It is acknowledged that excessive reactive oxygen species (ROS) level attributes greatly to the compromised developmental potential of oocytes matured in vitro. Although agents were applied to alleviate ROS levels, results were varied because of the distinct antioxidative activity and cell toxicity. Leonurine (LEO), extracted from the natural Chinese herb motherwort, is considered to be a potent free radical scavenger. Yet, it is undetermined whether LEO is benefit for oocyte development during in vitro maturation (IVM). In the present study, the effect of LEO on the quality of bovine oocyte as well as the underlying mechanism was investigated. We found that maturation rate (P < 0.01), subsequent blastocyst formation rate (P < 0.05), and the total blastocyst cell number (P < 0.05) after parthenogenetic activation were significantly increased in the group treated with 20 µM LEO. Moreover, a dramatic decline in ROS (P < 0.01), decreased lipid content (P < 0.01), elevated MMP level (P < 0.05), increased ATP content (P < 0.05), and reduced mitochondrial temperature (P < 0.01) were observed in oocytes treated with LEO. Furthermore, the expression level of anti-apoptotic protein BCL2 was significantly higher in LEO treated oocytes (P < 0.01), and the ratio of BAX/BCL2 was obvious decreased (P < 0.01). Finally, we found that LC3B intensity was significantly reduced (P < 0.05) while the rate of EdU positive nuclei was markedly increased (P < 0.05) in embryos derived from LEO-treated oocytes. Our results demonstrate that LEO exhibits a potent protective role in the acquisition of oocyte development capacity against oxidative stress during IVM, and provides a new solution for optimizing the in vitro culture system of bovine embryos.

Discovery and characterization of a novel cGAS covalent inhibitor for the treatment of inflammatory bowel disease.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, acts as a nucleotidyl transferase that catalyzes ATP and GTP to form cyclic GMP-AMP (cGAMP) and plays a critical role in innate immunity. Hyperactivation of cGAS-STING signaling contributes to hyperinflammatory responses. Therefore, cGAS is considered a promising target for the treatment of inflammatory diseases. Herein, we report the discovery and identification of several novel types of cGAS inhibitors by pyrophosphatase (PPiase)-coupled activity assays. Among these inhibitors, 1-(1-phenyl-3,4-dihydro-1H-pyrrolo[1,2-a]pyrazin-2-yl)prop-2-yn-1-one (compound 3) displayed the highest potency and selectivity at the cellular level. Compound 3 exhibited better inhibitory activity and pathway selectivity than RU.521, which is a selective cGAS inhibitor with anti-inflammatory effects in vitro and in vivo. Thermostability analysis, nuclear magnetic resonance and isothermal titration calorimetry assays confirmed that compound 3 directly binds to the cGAS protein. Mass spectrometry and mutation analysis revealed that compound 3 covalently binds to Cys419 of cGAS. Notably, compound 3 demonstrated promising therapeutic efficacy in a dextran sulfate sodium (DSS)-induced mouse colitis model. These results collectively suggest that compound 3 will be useful for understanding the biological function of cGAS and has the potential to be further developed for inflammatory disease therapies.

Discovery of ARF1-targeting inhibitor demethylzeylasteral as a potential agent against breast cancer.

Image 1.

Procyanidin B2 Protects Aged Oocytes Against Meiotic Defects Through Cortical Tension Modulation.

Defects in meiotic process are the main factors responsible for the decreased developmental competence in aged oocytes. Our recent research indicated that natural antioxidant procyanidin B2 (PCB2) promoted maturation progress in oocytes from diabetic mice. However, the effect of PCB2 on aging-induced chromosome abnormalities and the underlying mechanism have not been explored. Here, we found that PCB2 recovered aging-caused developmental arrest during meiotic maturation, germinal vesicle breakdown (GVBD) rate was significantly higher in aged oocytes treated with PCB2 (P < 0.05). Furthermore, we discovered that cortical mechanics were altered during aging process, cortical tension-related proteins were aberrantly expressed in aged oocytes (P < 0.001). PCB2 supplementation efficaciously antagonized aging-induced decreased cortical tension (P < 0.001). Moreover, PCB2 restored spindle morphology (P < 0.01), maintained proper chromosome alignment (P < 0.05), and dramatically reduced reactive oxygen species (ROS) level (P < 0.05) in aged oocytes. Collectively, our results reveal that PCB2 supplementation is a feasible approach to protect oocytes from reproductive aging, contributing to the improvement of oocytes quality.

[A clinical study of thunder-fire moxibustion combined with meibomian gland massage in treatment of meibomian gand dysfunction].

OBJECTIVE: To observe the effect of thunder-fire moxibustion combined with meibomian gland massage in improving meibomian gland dysfunction (MGD) and explore its mechanism. METHODS: Seventy-two MGD patients with 144 eyes in the Jinhua Hospital of Traditional Chinese Medicine from February 2019 to January 2021 were selected and randomly divided into an experimental group (n=36,72 eyes) and a control group (n=36, 72 eyes). Patients in the control group received 0.1% fluo-rometholone eye drops and 0.1% sodium hyaluronate eye drops, 1-2 drops per time, four times per day, and the meibomian glands were massaged once per day. Patients in the experimental group received additional thunder-fire moxibustion on the basis of the treatment of the control group, 10 cones per time, once per day. One month after treatment, meibomian gland function was assessed, and the levels of interleukin-6 (IL-6) and prostaglandin E2(PGE2) in tears were detected. RESULTS: After treatment, the scores of Ocular Surface Disease Index, meibomian hyperemia, meibomian gland opening, meibomian gland loss, and meibomian gland secretion function were lower than those before treatment in the two groups, and the scores of the experimental group were lower than those of the control group (P<0.05). After treatment, the tear break-up time and tear meniscus height were higher than those before treatment in the two groups, which were higher in the experimental group than those in the control group (P<0.05). The post-treatment levels of IL-6 and PGE2 were lower than those before treatment in the two groups, and the levels in the experimental group were lower than those in the control group (P<0.05). CONCLUSION: Thunder-fire moxibustion combined with meibomian gland massage can significantly improve the function of the meibomian glands and lower the levels of IL-6 and PGE2 in tears.

Discovery of the First-in-Class Agonist-Based SOS1 PROTACs Effective in Human Cancer Cells Harboring Various KRAS Mutations.

Regulating SOS1 functions may result in targeted pan-KRAS therapies. Small-molecule SOS1 inhibitors showed promising anticancer potential, and the most advanced inhibitor BI 1701963 is currently under phase I clinical studies. SOS1 agonists provide new opportunities to treat cancer; however, the underlying mechanisms still warrant investigation. We here report the discovery of the first SOS1 PROTACs designed uniquely by connecting a VHL ligand to the reported SOS1 agonist, ensuring that the observed inhibitory activity results from degraders. The best compound 9d induced SOS1 degradation in various KRAS-driven cancer cells and displayed superior antiproliferation activity compared to the agonist itself. Tumor xenograft study clearly showed the promising antitumor potency of 9d against human lung cancer. This study provides good evidence of using agonists to design SOS1 PROTACs and demonstrates that targeted SOS1 degradation represents an effective therapeutic strategy for overcoming KRAS-driven cancers.

Effects of hypoxia on DNA hydroxymethylase Tet methylcytosine dioxygenase 2 in a KG-1 human acute myeloid leukemia cell line and its mechanism.

Hypoxia is involved in the epigenetic modification of leukemia. As an important DNA hydroxymethylase and a tumor suppressor gene, the expression regulating mechanism of Tet methylcytosine dioxygenase 2 (TET2) remains unclear. The aim of the present study was to explore whether hypoxia and hypoxia-inducible factor 1α (HIF-1α) regulate TET2 gene expression and its demethylation function in acute myeloid leukemia (AML). The human AML cell line KG-1 was used in the present study. The results demonstrated that hypoxia could increase proliferation, enhance metabolism and inhibit apoptosis in KG-1 cells, as detected by the cell counting kit-8 assay, lactate dehydrogenase assay and Annexin V-FITC/propidium iodide staining, respectively. Hypoxia reduced the genome methylation status in KG-1 cells detected using 5-methylcytosine and 5-hydroxymethylcytosine detection kits. In addition, HIF-1α overexpression increased TET2 expression, 5-hmC level and cyclin-dependent kinase inhibitor 2B [p15(INK4B)] gene demethylation compared with the HIF-1α non-overexpression group in KG-1 cells detected by reverse transcription-quantitative PCR, western blotting, 5-hydroxymethylcytosine detection kits and methylation-specific PCR, respectively. The inhibition of HIF-1α by inhibitor YC-1 reduced demethylation in KG-1 cells by decreasing TET2 expression. It was also revealed that HIF-1α could enhance TET2 transcriptional activity by binding to the hypoxia response element of the TET2 gene promoter region using chromatin immunoprecipitation and luciferase reporter gene assays. TET2 may be a potential target gene regulated by HIF-1α. Hypoxia was demonstrated to regulate the expression of TET2 by HIF-1α, which in turn affected the methylation and expression of downstream target genes and served a role in the occurrence and progression of leukemia. In the present study, the association between hypoxia metabolism and epigenetic regulation in AML was investigated and the findings provided a new idea and experimental basis for the diagnosis and treatment of hematologic malignancies.

[Effect on the pain and tear film stability in patients after pterygium excision treated with intradermal needling].

OBJECTIVE: To analyze the effects of intradermal needling for pain and tear film stability in patients after pterygium excision. METHODS: A total of 76 patients (98 affected eyes) with primary pterygium were randomly divided into an observation group (38 cases, 53 affected eyes) and a control group (38 cases, 45 affected eyes).In the control group, only pterygium resection was performed, in the observation group, intradermal needling after pterygium resection was applied at Cuanzhu (BL 2), Yuyao (EX-HN 4), Taiyang (EX-HN 5), Sibai (ST 2), Hegu (LI 4), removed after 24 h and changed three times a week. The pain level of 3 days after surgery, dry eye symptoms, the basic tear secretion test (Schirmer-Ⅰ), and the tear-break time (BUT) changes before surgery, 2 weeks after surgery and 4 weeks after surgery were compared between the two groups, and the clinical efficacy was evaluated. RESULTS: The pain level of 3 days after surgery in the observation group was significantly lower than that in the control group (P<0.05). The dry eye symptom scores at 2 weeks and 4 weeks after surgery in the two groups were significantly lower than those before surgery (all P<0.05), and the dry eye symptom scores in the observation group were significantly lower than those in the control group (both P<0.05). The Schirmer-Ⅰ test at 2 weeks and 4 weeks after surgery was significantly prolonged than that before surgery(all P<0.05), and the Schirmer-Ⅰ test in the observation group was significantly longer than that in the control group (both P<0.05). The BUT at 2 weeks and 4 weeks after surgery in the two groups was significantly longer than that before surgery (all P<0.05), and the BUT in the observation group was significantly longer than that in the control group (both P<0.05). The total effective rate in the observation group was 89.5% (34/38), which was higher than 71.1% (27/38) in the control group (P<0.05). CONCLUSION: Intradermal needling can effectively reduce the pain level of patients after pterygium resection, improve dry eye symptoms, promote the secretion of tears and improve the tear film stability.

[Establishment and verification of dose effect relation curves between T cell receptor gene mutation frequency and ionizing radiation dose].

OBJECTIVE: To establish the dose-effect curve between TCR MF and ionizing radiation. METHODS: Peripheral lymphocytes were collected from 8 healthy adults (4 males and 4 females) and cultured in vitro with 12 well culture plates. They were stimulated by PHA-P and IL-2 after exposed to different doses of irradiation (0.00 - 8.00 Gy) and cultured for 7 d. The dose-effect curve was established after measuring TCR MF using flow cytometry. Also, using the same method, we separated and cultured the peripheral lymphocytes collected from 16 radiotherapy cancer patients, whose radiation styles and doses were different, and then measured TCR MF to estimate the whole equivalent dose of radiotherapy patients through the dose-effect curve. Peripheral blood was collected and cultured, chromosome aberration (dicentric and ring) was determined under microscope to estimate irradiation dose. RESULTS: The relationship of dose-effect between the TCR MF and ionizing radiation (0.00 - 8.00 Gy) was well, the curve of large dose group (2.00 - 8.00 Gy), low dose group (0.00 - 1.00 Gy) and 0.00 - 8.00 Gy dose group were met with the quadratic polynomial model, the equation was TCR MF = -32.8579 + 20.5436D + 0.6341D(2), TCR MF = 1.796 + 0.017D + 5.155D(2) and TCR MF = -0.6229 + 6.305D + 0.6919D(2), respectively. D was the radiation dose (Gy). Using the established curve and the chromosome aberration method to estimate the systemic exposure dosage, the average relative deviation was 16.8%. CONCLUSION: The curve established by the TCR gene mutation analysis technology can be applied to exposure dose estimation of victims in ionization radiation accidents.

The reversibility of neurofilaments decline induced by 2,5-hexanedione in rat nerve tissues.

To investigate the reversibility of the neuropathy induced by 2,5-HD, adult male rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week) for 2, 4, and 8 weeks, respectively. After stopping HD exposure, half of 8-week treated animals were allowed to naturally recover for 16 weeks. The relative levels of NF-H, NF-M, and NF-L in spinal cords and sciatic nerves of rats were determined by immunoblotting during the HD neuropathy. The results showed that NFs content in nerve tissues demonstrated a progressive decline as the intoxication continued. Furthermore, after a recovery of 16 weeks, the levels of three NF subunits in spinal cords of treated rats returned to normal while those in sciatic nerves displayed an inconsistent reversal. Among them, the level of NF-H in sciatic nerves returned to normal completely, and NF-L also showed a significant improvement, whereas NF-M did not demonstrate an obvious reversal. These findings suggest that HD-induced NFs decline is at least partially irreversible within the time frame of this study, which might be associated with the incomplete recovery of neurological dysfunctions of HD-treated rats.

Time-dependent changes of lipid peroxidation and antioxidative status in nerve tissues of hens treated with tri-ortho-cresyl phosphate (TOCP).

Tri-ortho-cresyl phosphate (TOCP) could induce a delayed neurodegenerative condition known as organophosphorus easter-induced delayed neurotoxicity (OPIDN) in human beings and sensitive animals. However, the mechanisms of OPIDN remain unknown. This study investigated the time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues for elucidating the mechanism of OPIDN induced by TOCP. Adult hens were treated with TOCP by gavage at a single dosage of 750 mg/kg. TOCP was dissolved in corn oil and administered at 0.65 ml/kg. The control hens received an equivalent volume of corn oil by gavage. Hens were sacrificed after 0, 5, 10, 15 and 21 days of treatment and the cerebrum, spinal cord, sciatic nerve were dissected, homogenized and used for the determination of lipid peroxidation and antioxidative status. The results showed that treatment with TOCP increased lipid peroxidation and reduced the antioxidative status in cerebrum, spinal cord and sciatic nerve. The levels of MDA increased by 33% (P<0.01) in cerebrum on 5th day after TOCP treatment and at clinical sign score of 1-2, and increased respectively by 32% and 15% (P<0.01) in spinal cord and sciatic nerve on 10th day after TOCP treatment and at clinical sign score of 3-4. Further changes of MDA were also observed after 15 and 21 days post-dosing and at clinical sign score of 5-6 and 7-8. There is a decrease in the activities of SOD, GSH-Px, GR, anti-ROS, and GSH content in cerebrum, spinal cord and sciatic nerve of hens after 5, 10, 15 and 21 days post-dosing and at clinical sign score of 1-2, 3-4, 5-6 and 7-8. Thus, OPIDN induced by TOCP was associated with elevation of lipid peroxidation and reduction of antioxidative status, and the time-dependent changes of these indexes in hens nerve tissues occurred. Sciatic nerve was the main target tissue and MDA was most sensitive among all indexes. The time-dependent and tissue specific changes of lipid peroxidation and antioxidative status in cerebrum, spinal cord and sciatic nerve suggest that ROS and concomitant lipid peroxidation, at least in part, are involved in the toxic effects of TOCP on nerve tissues and that oxidative stress may play a role in the occurrence and development of OPIDN induced by TOCP.