Dynamic Analysis of the Biochemical Changes in Rats with Polycystic Ovary Syndrome (PCOS) Using Urinary 1H NMR-Based Metabonomics.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disease that causes reproductive abnormalities in fertile women. It is closely related to the persistent anovulatory, insulin resistance, and high androgen. However, the molecular mechanisms underlying the pathological development of PCOS are still unclear. In this study, we aimed to explore the distinctive metabolic patterns in insulin combined with human chorionic gonadotrophin induced PCOS. The dynamic changes of endogenous metabolites in the development of PCOS were studied using untargeted metabolomic approaches based on nuclear magnetic resonance. The results showed that the degree of PCOS disorder metabolites at different periods was not exactly the same. Twelve significantly differential endogenous metabolites from different time points were selected as potential biomarkers relate to pathological process of PCOS. Among them, six metabolites showed a good diagnostic accuracy with PCOS model. The arginine and proline metabolic pathway was considered as one of the most crucial pathways that affects occurrence and development of PCOS. In addition, IRS-1, Akt, PI3K, IκB, and NF-κB (p65) were significantly changed in the ovary tissue of PCOS rats, which revealed that the IRS-1-PI3K/Akt and NF-κB signal pathway may be involved in the development of PCOS. This study demonstrated that metabolomic analysis is a powerful tool for providing novel insight into understanding the pathogenesis of PCOS and provide a basic reference for the diagnosis of PCOS at the onset stage.

Expression of Bcl-2, Bax and Caspase-3 in nerve tissues of rats chronically exposed to 2,5-hexanedione.

Occupational exposure and experimental intoxication with n-hexane or its metabolite 2,5-hexanedione (HD) produce a central-peripheral neuropathy. However, the mechanism remains unknown. We hypothesized that HD affected the expression of Bcl-2, Bax and Caspase-3 in the central nervous system (CNS) and the peripheral nervous system (PNS). Male adult Wistar rats were administered by intraperitoneal injection at a dosage of 200 or 400 mg/kg HD, five days per week for 8 weeks. Samples of the cerebral cortex, cerebellum, spinal cord and sciatic nerves were collected and examined for Bcl-2, Bax and Caspase-3 expression using Western blotting. Subchronic exposure to HD resulted in significantly increased expression of both anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax and Caspase-3 in cerebral cortex and cerebellum, which exhibited a dose-dependent pattern. Though little change was detected in spinal cord, our results showed that the expression of Bcl-2, Bax and Caspase-3 was markedly enhanced in the sciatic nerves. These findings suggested that the changes of apoptosis-related protein level in rat nerve tissues were associated with the intoxication of HD, which might be involved in early molecular regulatory mechanism of apoptosis in the HD-induced neuropathy.

Expression changes of neurofilament subunits in the central nervous system of hens treated with tri-ortho-cresyl phosphate (TOCP).

Tri-ortho-cresyl phosphate (TOCP) could induce degeneration of long, large diameter axons within the central and peripheral nervous system of susceptible species including human being and hens, which is referred to as organophosphorus-ester induced delayed neuropathy (OPIDN). The mechanisms involved are not understood. Neuropathologic observations suggested that neurofilament subunits (NFs) could be a main target of TOCP in the peripheral nervous system. Our previous study also showed that NFs in protein levels significantly decreased in sciatic nerves of hens treated with TOCP. In this study, to determine whether the decrement of NFs proteins in sciatic nerves was due to reductions in NF gene expression or protein degradation, hens were treated with a single dose of 750 mg/kg body weight TOCP by gavage, and sacrificed on 21 day post-exposure. Cerebral cortexes and spinal cords were sampled. Transcriptional changes of NFs including high molecular weight neurofilament (NF-H), middle molecular weight neurofilament (NF-M), low molecular weight neurofilament (NF-L), and glyceraldehydes-3-phoaphate dehydrogenase (GAPDH) as inner inference in cerebral cortexes and spinal cords were analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results showed that all of three NFs mRNA in cerebral cortexes down-regulated significantly. However, in spinal cords, there was only NF-M decreased, both of NF-H and NF-L kept unaffected. The protein levels of NFs in pellet and supernatant fractions of cerebral cortexes and spinal cords were also determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. We noticed that all NFs protein declined in pellet of cerebral cortexes, but NF-M reduction was not significant compared with that of control hens. NF-H and NF-M proteins in supernatant of cerebral cortexes exhibited significant increase, while NF-L level showed remarkable decline. In spinal cords, apart from NF-L in pellet were significantly increased, both of NF-H and NF-M in pellet and supernatant, as well as NF-L in supernatant fractions were manifested dramatic reduction compared with the pattern of control. The quantitative analyses revealed that the change magnitude in protein levels was much greater than that in mRNA levels in hens' central nervous system after TOCP administration. These findings suggest that the NFs disturbance in protein levels is closely associated with the decreases in sciatic nerves observed in our previous work after TOCP exposure, rather than that in mRNA levels, and the NFs alterations in protein levels may be one of the responsible factors for the OPIDN.

Effect of subchronic exposure to acrylamide induced on the expression of bcl-2, bax and caspase-3 in the rat nervous system.

Occupational exposure and experimental intoxication with acrylamide (ACR) produce a neuropathy characterized by nerve degeneration. We hypothesize that ACR differentially affects the expression of bcl-2, bax and caspase-3 in the central nervous system (CNS) and the peripheral nervous system (PNS) tissue. Male adult Wistar rats were given ACR (20, 40 mg/kg i.p. 3 days/week) for 8 weeks. Samples of the cerebral cortex, cerebellum, spinal cord and sciatic nerves were collected and examined for bcl-2, bax and caspase-3 expression using Western blotting. Subchronic exposure to ACR reduced cortical bcl-2 expression in the low dose, increased it in the high dose; the change of bcl-2 expression in the spinal cord and cerebellum followed the same pattern as that described in the cerebral cortex; there was no significant change in the expression of bax in the cerebral cortex and the spinal cord, however, in the cerebellum the change of bax expression and bcl-2 expression is just the reverse. Thus, the bcl-2/bax ratio of the CNS tissue was affected by exposure to ACR, it decreased in the low dose group and increased in the high group. Compared to control, densitometric analysis showed that in the sciatic nerves the expression of bcl-2 and bax expression was markedly increased following ACR administration. The expression of inactive isoforms (32 kDa) of caspase-3 was not altered in the cortices of ACR-treated rats, but increased in their spinal cords and sciatic nerves. Thus, subchronic exposure to ACR affected the expression of death-related proteins in the CNS and PNS tissue, which indicate there is the early molecular regulatory mechanism of apoptosis in the neuropathy induced by ACR.

Acrylamide-induced changes in the neurofilament protein of rat cerebrum fractions.

Acrylamide (ACR) is known to produce central-peripheral distal axonopathy, which is characterized by distal swellings and secondary degeneration both in experimental animals and human. Ultrastructurally, excessive accumulation of neurofilaments (NFs) in the distal swollen axon is a major pathological hallmark. However, the mechanisms of ACR axonopathy remain unknown. Twenty seven male Wistar rats were randomly divided into three groups. Lower and higher ACR groups were received 20 and 40 mg/kg ACR by i.p. injection respectively. The control group received physiological saline. All rats were sacrificed after 8 weeks of treatment and their cerebrums were dissected, homogenized and used for the determination of the NF proteins. In general, the levels of light NF (NF-L) and medium NF (NF-M) subunits increased consistently in the supernatant, whereas they decreased consistently in the pellet from rats treated with ACR. Compared to that of the control group, the levels of NF-L increased respectively by 104% and 45% (P<0.01) in the supernatant and decreased by 16% and 11% (P<0.01) in the pellet of rat cerebrums in lower and higher groups. The enhancement of NF-M was 76% and 147% (P<0.05, P<0.01) in supernatant, and the reduction was 26% and 36% (P<0.01) in pellet in lower and higher group respectively. The heavy NF (NF-H) level changed slightly. The present results suggested that the change of NF-L and NF-M levels in cerebrum might be relevant to the mechanisms of the neurofilamentous axonopathies induced by ACR.