[Chronic Injury of Mice Bone Marrow Multipotent Hematopoietic Progenitor Cells Induced by Ionizing Radiation].

OBJECTIVE: To explore the chronic injury and its possible mechanism of ionizing radiation on multipotent hematopoietic progenitor cells (MPPs) by determining the related indicators of MPPs in bone marrow of mice post-radiation. METHODS: Sixteen C57BL/6 adult mice were randomly divided into normal control and irradiation groups, 8 mice in each group. The mice in irradiation group were exposed to 6 Gy X-ray. The proportion of bone marrow MPPs, their apoptosis and proliferation 2 months after irradiation were detected by flow cytometry. Mitochondrial activity and levels of reactive oxygen species (ROS) in each MPPs population were detected by Mitotracker Red and DCFDA probes, and the senescent state of MPPs in the bone marrow was analyzed. RESULTS: Ionizing radiation could reduce the proportion of MPPs in mouse bone marrow. The proportions and numbers of MPP1, MPP3 and MPP4 in the bone marrow were significantly decreased after whole-body irradiation with 6 Gy X-ray (P<0.05). In addition, radiation significantly reduced the colony-forming capacity of MPPs in bone marrow (P<0.05), the proportions of apoptotic cells in the MPP1 and MPP4 cell populations increased significantly in the bone marrow (P<0.05). The activity of mitochondria was significantly reduced in the bone marrow MPP2, MPP3 and MPP4 cell populations compared with that of the control group (P<0.05). It was also found that the radiation could significantly increase the ROS levels of MPPs in bone marrow, and the content of ROS in the MPP2, MPP3 and MPP4 cell population of the bone marrow was significantly increased(P<0.05). The senescent cells ratios of MPP1, MPP3 and MPP4 cells in the bone marrow after irradiation were significantly higher than those in the control group (P<0.05). CONCLUSION: Ionizing radiation can cause chronic MPPs damage in mice, which is closely associated with persistent oxidative stress, cells apoptosis, and cellular senescence.

Correlation between the expressions of circular RNAs in peripheral venous blood and clinicopathological features in hepatocellular carcinoma.

BACKGROUND: Recent studies have reported that circular RNAs (circRNAs) are involved in the development of hepatocellular carcinoma (HCC). This study evaluated the expression of preoperative peripheral venous blood circRNAs in HCC patients and their predictive ability for microvascular invasion (MVI). METHODS: Seven circRNAs (circMTO1, circ-10720, circZKSCAN1, cSMARCA5, circHIPK3, circSETD3 and ciRS-7) were screened from the literature as circRNAs with reported biological functions in HCC. The expression levels of seven circRNAs in preoperative blood samples and HCC tissues were detected by quantitative reverse transcription polymerase chain reaction. The correlations between the circRNA expressions in blood and the clinicopathological factors of HCC patients were analyzed. The risk factors of MVI were analyzed by univariate and multivariate logistic regression. The functional role of circSETD3 in cell migration and invasion was evaluated by wound healing and Transwell assays in vitro. RESULTS: The expressions of all seven circRNAs were measured in peripheral venous blood samples. The venous expression levels of circHIPK3 and circMTO1 were significantly associated with gender, while circ-10720 and circMTO1 levels were significantly correlated with gross vascular invasion. Furthermore, circMTO1 and cSMARCA5 levels were significantly associated with alpha-fetoprotein level and ciRS-7 was significantly associated with satellite nodules. Importantly, low venous circSETD3 expression was significantly associated with prothrombin induced by vitamin K absence or antagonist-II (PIVKA-II) level, MVI, gross vascular invasion, and liver capsule. Furthermore, venous circSETD3 expression had predictive ability for MVI. Knockdown of circSETD3 promoted cell invasion and metastasis in vitro. CONCLUSIONS: CircRNAs were stably present in peripheral venous blood and associated with multiple clinicopathological characteristics of HCC patients. Venous circSETD3 was an independent risk factor of MVI and shows ability to predict MVI in HCC patients before surgery.

[Mechanism of trichosanthin inducing apoptosis of mouse prostatic cancer RM-1 cells in vitro].

OBJECTIVE: To study the inducing effect of trichosanthin on the apoptosis of mouse prostatic cancer RM-1 cells and its mechanism. METHODS: MTT assay was used to identify the effect of trichosanthin on RM-1 cells in vitro. The cells apoptosis was detected by Hoechest 33258 fluorescent staining and flow cytometry. The levels of bax proteins were detected by western blot analysis as well as their apoptosis rate. RESULTS: The IC50 value of trichosanthin for RM-1 cell was 117.32 mg/L. Cell cycle was analysised by flow cytometry, trichosanthin induced an arrest in G0/G1 phase. Hoechest 33258 fluorescent staining showed typical nucleolus changes in apoptosis cells. Expression of bax was up-regulated at protein levels in a time-dependent manner. CONCLUSION: Trichosanthin can induce apoptosis in RM-1 cells. The induction of apoptosis is a very important mechanism of trichosanthin to inhibit prostatic cancer.

Impaired reproduction in transgenic mice overexpressing Gamma-aminobutyric acid transporter I (GAT1).

It is well documented that g-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis, and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.

Identification of glutamate transporters and receptors in mouse testis.

AIM: To investigate the presence of glutamate transporters and receptors in mouse testis. METHODS: Glutamate uptake analysis was performed to study the function of glutamate transporters in mouse testis. Comparative RT-PCR technique and sequencing analysis were used to study the expression of glutamate receptors and transporters in mouse testis. RESULTS: Mouse testis possessed glutamate uptake capacity with sodium-dependence. Vmax value of glutamate uptake was (1.60 +/- 0.21) pmol/min per mg protein and Km value of glutamate uptake was (11.0+/-1.6) micromol/L in mouse testis according to saturation analysis. Furthermore, the uptake activity could be inhibited by DHK (GLT1 selective inhibitor) and THA (glutamate uptake inhibitor). In addition, RT-PCR results revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6 and KA2) were expressed in mouse testis. CONCLUSION: Glutamate transporters and receptors do exist in mouse testis.

Identification of glutamate receptors and transporters in mouse and human sperm.

gamma-Aminobutyric acid (GABA) and glutamate (Glu) are considered as the predominant inhibitory and excitatory neurotransmitters in mammalian central nervous systems (CNS), respectively. The presence of the GABA system and metabotropic glutamate receptors in sperm prompted us to explore the existence of ionotropic glutamate receptors and glutamate transporters in sperm. Immunofluorescent analysis was used to investigate the existence and location of glutamate, glutamate receptor (NR2B), and glutamate transporter (GLT1) in mouse and human sperm. Our present results showed that NR2B was located in the midpiece of sperm, whereas GLT1 mainly existed in the head. Moreover, glutamate uptake activity was detected in mouse sperm and it could be blocked by dihydrokainic acid (DHK, GLT1-selective inhibitor) and DL-threo-beta-hydroxyaspartic acid (THA, nonselective inhibitor). In addition, reverse transcription-polymerase chain reaction technique and sequencing analysis revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6, and KA2) existed in mouse sperm as well as in human sperm. The present findings are the first direct evidence for the existence of ionotropic glutamate receptors and glutamate transporters in sperm. It also indicates that, in sperm, glutamate receptors and transporters might have functions other than neurotransmission.