Hydrogen-rich saline ameliorates hippocampal neuron apoptosis through up-regulating the expression of cystathionine ß-synthase (CBS) after cerebral ischemia- reperfusion in rats.

OBJECTIVES: This study aimed to evaluate the potential role of hydrogen in rats after cerebral ischemic/reperfusion (I/R) injury. MATERIALS AND METHODS: The experimental samples were composed of sham group, model group of rats that received middle cerebral artery occlusion (MCAO) for 2 hr followed by reperfusion for 24 hr, and the hydrogen saline group treated by hydro¬gen-rich saline (1 ml/kg) after MCAO. Hydrogen sulfide (H2S), S100-ßprotein (S100-ß), and neuron-specific enolase (NSE) levels were measured; the levels of malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD) were detected; the histologic structure and apoptotic cells of hippocampus were observed; the expressions of cystathionine ß-synthase (CBS), nuclear factor erythroid 2-related factor 2 (Nrf2), and hemeoxygenase-1 (HO-1) were measured. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test. RESULTS: Our results showed that hydrogen up-regulated H2S levels via promoting the expression of CBS in the hippocampus, and its treatment alleviated oxidative stress via activating the expression of Nrf2 and HO-1, and then cell apoptosis reduced, furthermore, brain function improved by down-regulating the levels of S100-ßand NSE. CONCLUSION: This study showed that hydrogen-rich saline ameliorates cell injury through up-regulating the expression of CBS in the hippocampus after cerebral ischemia reperfusion (I/R) in rats, this provides new experimental evidence for the treatment of stroke with hydrogen saline.

Endosulfan induces endothelial inflammation and dysfunction via IRE1α/NF-κB signaling pathway.

Cardiovascular diseases are related to vascular endothelial cell injury; our previous studies showed that endosulfan could cause hypercoagulation of blood by inducing endothelial cell injury. To clarify the mechanism of it, we treated human umbilical vein endothelial cells (HUVECs) with 0, 1, 5, and 10 µg/mL endosulfan, while in the inhibition groups, reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC, 3 mmol) and endoplasmic reticulum (ER) stress inhibitor (STF-083010, 10 µmol) were incubated prior to endosulfan. The results showed that endosulfan could induce inflammatory response and dysfunction by increasing the release of inflammatory cytokines such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1) and endothelin-1 (ET-1), and inducing ROS production in HUVECs. We also found that endosulfan could cause ER damage, remarkably increase the expressions of inositol-requiring enzyme 1α (IRE1α), phosphorylated IRE1α (p-IRE1α), GRP78, XBP1, nuclear factor-kappa B (NF-κB), and phosphorylated NF-κB (p-NF-κB) in HUVECs. The presence of NAC antagonized the ROS production, expressions of IRE1α and p-IRE1α; however, STF-083010 could decrease the expression levels of GRP78, XBP1, NF-κB, and p-NF-κB and attenuate IL-1ß, IL-6, TNF-α, VCAM-1, and ET-1 release induced by endosulfan. These results demonstrated that endosulfan-induced endothelial inflammation and dysfunction through the IRE1α/NF-κB signaling pathway may be triggered by oxidative stress. The study provided experimental basis for the correlation between environmental pollutants (endosulfan) and cardiovascular diseases.

Adiponectin alleviates blood hypercoagulability via inhibiting endothelial cell apoptosis induced by oxidative stress in septic rats.

OBJECTIVES: The purpose of this study was to detect the protective effects of adiponectin on coagulation dysfunction and its mechanism in sepsis of rats. MATERIALS AND METHODS: The experimental samples were composed of sham group, model group that was underwent cecal ligation and puncture (CLP) and three adiponectin treatment groups that treated by adiponectin with different dose (72 µg/kg, 96 µg/kg and 120 µg/kg) after CLP. The prothrombin time (PT), activated partial thromboplastin time (APTT) was measured, respectively, the level of malondialdehyde (MDA), tissue factor (TF), activated coagulation factor VIIa and Xa, p-selectin were detected, the histology structure of vascular was observed, the expressions of Caspase 9, Caspase 3, Bax, Bcl-2 and vWF in vascular were measured. RESULTS: The results demonstrated that adiponectin treatment lengthened PT and APTT, reduced the expression of MDA, TF, activated coagulation factor VIIa, Xa and p-selectin in plasma of septic rats. Additionally, adiponectin treatment alleviated endothelial cell apoptosis and oxidative stress, down-regulated the levels of Caspase 3, Caspase 9, Bax, Bcl-2 and vWF in vascular. CONCLUSION: These findings suggest that adiponectin treatment might be a promising therapeutic strategy for relieving septic endothelial cell injury and coagulation dysfunction via inhibiting endothelial cell apoptosis in septic rats.

Adiponectin is protective against endoplasmic reticulum stress-induced apoptosis of endothelial cells in sepsis.

Endoplasmic reticulum (ER) stress is a critical molecular mechanism involved in the pathogenesis of sepsis. Hence, strategies for alleviating this stress may be essential for preventing cardiovascular injuries under sepsis. Adiponectin is secreted by adipocytes and its levels are decreased in sepsis. The purpose of this study was to investigate the protective effects of adiponectin treatment on endothelial cells and its mechanism. Male Wistar rats underwent cecal ligation and puncture (CLP) before being treated with adiponectin (72 and 120 µg/kg). The levels of malondialdehyde (MDA) in plasma, histological structure, and apoptosis of endothelial cells were evaluated. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with adiponectin at 10 and 20 µg/mL for 24 h after stimulation by lipopolysaccharide (LPS). The levels of reactive oxygen species (ROS), ultrastructure, rate of apoptosis, the expression of inositol-requiring enzyme 1α (IRE1α) protein, and its downstream molecules (78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12) were detected. The results showed that the levels of MDA and ROS induced by CLP or LPS stimulation were increased. Furthermore, endothelial cell apoptosis was increased under sepsis. The IRE1α pathway was initiated, as evidenced by activated IRE1α, increased GRP78, and up-regulated CHOP and caspase-12 in HUVECs. Following treatment with adiponectin, the number of apoptotic endothelial cells was markedly decreased. These findings demonstrated that treatment with adiponectin decreased apoptosis of endothelial cells caused by sepsis by attenuating the ER stress IRE1α pathway activated by oxidative stress.

Tetramethylpyrazine ameliorates experimental autoimmune encephalomyelitis by modulating the inflammatory response.

Multiple sclerosis (MS) is a disabling inflammatory and demyelinating disorder of the central nervous system. Tetramethylpyrazine (TMP) has been demonstrated to ameliorate cerebral ischemic injury and spinal cord injury by inhibiting inflammatory cell activation and pro-inflammatory cytokine production. However, the effects of TMP on MS have not been studied. In this study, we evaluated the effects of TMP on the inflammatory response in experimental autoimmune encephalomyelitis (EAE), which is an animal model of MS. TMP (30 mg/kg) treatment significantly reduced the expression levels of NLR Family, Pyrin Domain-Containing 3 Protein inflammasome and caspase-1and decreased inflammatory infiltration and glial activation. Moreover, TMP (30 mg/kg) suppressed the expression of pro-inflammatory cytokines (interleukin-18 [IL-18] and IL-17) and promoted the expression of an anti-inflammatory cytokine (IL-10). The reduced inflammatory response resulted in improvement in clinical scores and decreased demyelination in EAE mice. Therefore, our results demonstrate that TMP (30 mg/kg) improved functional recovery in part by reducing inflammation in EAE mice. TMP may be a potential therapeutic agent for MS therapy.

Adiponectin is protective against endoplasmic reticulum stress-induced apoptosis of endothelial cells in sepsis

Endoplasmic reticulum (ER) stress is a critical molecular mechanism involved in the pathogenesis of sepsis. Hence, strategies for alleviating this stress may be essential for preventing cardiovascular injuries under sepsis. Adiponectin is secreted by adipocytes and its levels are decreased in sepsis. The purpose of this study was to investigate the protective effects of adiponectin treatment on endothelial cells and its mechanism. Male Wistar rats underwent cecal ligation and puncture (CLP) before being treated with adiponectin (72 and 120 μg/kg). The levels of malondialdehyde (MDA) in plasma, histological structure, and apoptosis of endothelial cells were evaluated. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with adiponectin at 10 and 20 μg/mL for 24 h after stimulation by lipopolysaccharide (LPS). The levels of reactive oxygen species (ROS), ultrastructure, rate of apoptosis, the expression of inositol-requiring enzyme 1α (IRE1α) protein, and its downstream molecules (78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12) were detected. The results showed that the levels of MDA and ROS induced by CLP or LPS stimulation were increased. Furthermore, endothelial cell apoptosis was increased under sepsis. The IRE1α pathway was initiated, as evidenced by activated IRE1α, increased GRP78, and up-regulated CHOP and caspase-12 in HUVECs. Following treatment with adiponectin, the number of apoptotic endothelial cells was markedly decreased. These findings demonstrated that treatment with adiponectin decreased apoptosis of endothelial cells caused by sepsis by attenuating the ER stress IRE1α pathway activated by oxidative stress.

Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.

Retinoic acid amide inhibits JAK/STAT pathway in lung cancer which leads to apoptosis.

Small cell lung cancer (SCLC) accounts for 12 to 16% of lung neoplasms and has a high rate of metastasis. The present study demonstrates the antiproliferative effect of retinoic acid amide in vitro and in vivo against human lung cancer cells. The results from MTT assay showed a significant growth inhibition of six tested lung cancer cell lines and inhibition of clonogenic growth at 30 µM. Retinoic acid amide also leads to G2/M-phase cell cycle arrest and apoptosis of lung cancer cells. It caused inhibition of JAK2, STAT3, and STAT5, increased the level of p21WAF1, and decreased cyclin A, cyclin B1, and Bcl-XL expression. Retinoic acid amide exhibited a synergistic effect on antiproliferative effects of methotrexate in lung cancer cells. In lung tumor xenografts, the tumor volume was decreased by 82.4% compared to controls. The retinoic acid amide-treated tumors showed inhibition of JAK2/STAT3 activation and Bcl-XL expression. There was also increase in expression of caspase-3 and caspase-9 in tumors on treatment with retinoic acid amide. Thus, retinoic acid amide exhibits promising antiproliferative effects against human lung cancer cells in vitro and in vivo and enhances the antiproliferative effect of methotrexate.

Changes in PDGF expression in spared dorsal root ganglia and associated spinal dorsal horns in cats subjected to partial dorsal root ganglionectomy.

Changes in the platelet derived growth factor (PDGF) in the spared dorsal root ganglia (DRG) and associated spinal dorsal horns were evaluated in cats subjected to unilateral removal of L1-L5 and L7-S2 DRG, sparing the L6 DRG. The number of PDGF immunopositive neurons and protein expression decreased significantly in the spared DRG and associated dorsal horns of the L3 and L6 cord segments at 3 days post-operation (dpo). It bottomed to the lowest level at 7 dpo in the DRG, then returned to the control level at 14 dpo; while in the L6 dorsal horn, it rapidly increased at 7 dpo and exceeded the control level at 14 dpo. This showed a significant upregulation in the spared DRG and associated spinal dorsal horns, especially in the L6 cord segment following a transient decrease. Meanwhile, a significant upregulation of PDGF mRNA was also seen in L6 DRG and L3 and L6 dorsal horns at 3 dpo. The upregulation of the endogenous PDGF in the said structures indicated a potential role of this factor in spinal cord plasticity after partial dorsal root ganglia removal in cats.

Effects of electro-acupuncture on CNTF expression in spared dorsal root ganglion and the associated spinal lamina II and nucleus dorsalis following adjacent dorsal root ganglionectomies in cats.

It is well known that plasticity occurs in deafferented spinal cord, and that electro-acupuncture (EA) could promote functional restoration. The underlying mechanism is, however, unknown. Ciliary neurotrophic factor (CNTF) plays a crucial role in neurite outgrowth and neuronal survival both in vivo and in vitro, and its expression might explain some of the mechanism. In this study, we investigated the effects of EA on CNTF expression in the spared L(6) dorsal root ganglion (DRG), and spinal lamina II at spinal segments L(3) and L(6) as well as nucleus dorsalis (ND) of L(3) spinal segment following removal of L(1)-L(5) and L(7)-S(2) (DRG) in the cat. After ganglionectomies, the total and small-to-medium-sized numbers of immunoreactive neurons decreased at 3 dpo, and returned to the sham-operated level as early as 7 dpo. After EA, immunoreactive neurons in L(6) DRG noticeably increased at 7 dpo, compared with the non-acupunctured group. Notable increase in the large neurons was seen at 14 dpo, while their numbers in L(3) and L(6) spinal cord segments significantly declined at 3 dpo. Those in L(3) segment did not reach the sham-operated level until 14 dpo, but their numbers in L(6) segment returned to the sham-operated level as early as 7 dpo. CNTF immunopositive neurons in the ND of L(3) segment returned to the sham-operated level at 14 dpo. After EA, their number significantly increased as early as 7 dpo in lamina II of L(6) segment, and as late as 14 dpo in ND of L(3) segment. Western blot analysis showed CNTF changes corresponding to those shown in immunohistochemical staining. It is concluded that CNTF expression was involved in the EA promoted plastic changes in L(6) DRG and the associated deafferented spinal lamina and ND.

[The effect of acupuncture and endogenous c-Fos, c-Jun on regeneration of neuronal dendrite of spared DRG in vitro following partial ganglionectomy].

OBJECTIVE: To explore the effect of acupuncture, endogenous c-Fos and c-Jun on the regeneration of neuronal dendrite of spared dorsal root ganglion (DRG) in vitro following partial ganglionectomy. METHODS: Five adult male cats were used in this experiment. Their bilateral L1-L5, L7-S2 DRG were removed, and L6 DRG were spared. Then unilaterally, two sets of acupoints (Zusanli(St. 36) and Xuanzhong(G. B. 39); Futu (St. 32) and Sanyinjiao (Sp. 6) located in the distribution area of spinal nerve L6) were electro-stimulated alternately 30 min everyday by electro-needling. Seven days after operation, bilateral L6 DRGs were taken out and were cultured respectively in vitro. Some cultured mediums of the acupuncture lateral wells were totally replaced by each corresponding antibody-cultured medium including respectively 100 ng/mL anti-c-Fos and anti-c-Jun antibody at the 24th hour and terminated after 7 days. The length of the neurite was measured by upside-down light microscopy. Then, cultured cells were stained by the immunohistochemistry ABC method. Data were analyzed by One-way ANOVA and q test. RESULTS: Immunocytochemical staining revealed that over 95% cells were NSE positive cells which were the typical neuron of DRG in vitro. On the 7th day, the average neurite length of the spared DRG group, the anti-c-Fos antibody and the anti-c-Jun antibody group were shorter than that of the acupuncture group (P < 0.05); the average neurite length of the two antibody groups were longer than that of the spared DRG group (P < 0.05). CONCLUSION: These results indicate that acupuncture, endogenous c-Fos and c-Jun probably promote regeneration of neuronal dendrite of spared DRG in vitro.

[Effect of partial ganglionectomy and acupuncture on culturing spared DRG in vitro].

OBJECTIVE: To explore the effect of partial dorsal root rhizotomy and Acup on culturing dorsal root ganglion(DRG) in vitro. METHODS: Ten adult cats were divided into 2 groups: normal control group; Acup spared DRG 7 d group, in which bilateral L1-L5, L7-S2 DRG were removed; and L6DRG were spared; then unilaterally two sets of acupoints [Zusanlily (St. 36) and Xuanzhong (G. B. 39): Futu (St. 32) and Sanyinjiao (Sp. 6) located in the distribution area of spinal nerve L6] were electro-stimulated alternatively 30 min everyday by electro-needling. Five cats were used in every group. Bilateral L6 DRGs of every group were taken out on the condition of asepsis and were cultured respectively in vitro. Cultures were terminated after day 7. Then the cultured cells were stained under the same condition using specific NSE (1 : 200) antibody, a neuron-specific marker, by the immunohistochemistry ABC method. The neurite length was measured by micro-measured ruler in upside-down light microscope on the 1st, 3rd, 5th, 7th day. RESULTS: Immunocytochemical staining revealed that over 95% cells were NSE positive cells which were the typical neuron of DRG in vitro; on the 1st, 3rd, 5th, 7th day, the average neurite length of the normal group was shorter than that of the spared DRG group(P < 0. 05), and the spared DRG group's was shorter than the Acup group's at each time stage (P < 0.05). CONCLUSION: These results indicated that DRG had plasticity and acupuncture probably promoted the plasticity, which were probably in close relation with the spinal plasticity.

[Expression of CNTF and PDGF in spared dorsal root ganglion after partial dorsal root rhizotomy].

OBJECTIVE: To investigate the expression of ciliary neurotrophic factor (CNTF) and platelet derived growth factor (PDGF) in spared dorsal root ganglion (DRG) after partial dorsal root rhizotomy. METHODS: Twenty adult cats were divided into four groups. Five cats were kept intact in the control group. Fifteen cats were subjected to bilateral root rhizotomy, and on the 3rd day, 7th day and 14th day after operation, they were sacrificed as subjects in the three experiment groups respectively (n=5 per group) and their DRGs (L6) were taken. Immunochemical ABC method was used to detect the distribution of CNTF and PDGF-immunoreactivity neurons in those DRGs. The quantitative analysis was conducted to get the numbers of CNTF, PDGF-positive total, large, and medium-small sized neurons in each group. RESULTS: CNTF, PDGF-immunoreactants were distributed in large and medium-small sized neurons. The numbers of total and medium-small sized CNTF positive neurons were noted to be decreased on the 3rd day after operation (P<0.05), and no difference 7th and 14th days was seen when compared with control (P>0.05), but the large positive neurons showed no difference. The total and medium-small sized PDGF positive neurons were found decreased apparently on the 3rd day and 7th day, but there was no difference on the 14th day as compared with normal level. Large positive neurons displayed no change at every time. CONCLUSION: Partial dorsal root rhizotomy exerts different influence on the expression of CNTF and PDGF for different neurons in spared DRG.

[Changes in expression of SP in spinal lamina II following hemisected spinal cord injury in rats].

OBJECTIVE: To explore the changes in expression of Substance P (SP) in spinal lamina II at different time following hemisected spinal cord injury (hSCI). METHODS: Twenty Sprague-Dawley rats were randomly divided into intact group and hSCI group at days 3, 7 and 21. The spinal cords were hemisected between T13 and L1. The animals were sacrificed 3, 7, 21 days after operation. The L3 segments were taken out and sectioned continuously into sections (20 microm). The expression of SP was measured by immunohistochemical ABC method, and the number of SP positive varicosities in lamina II was counted. RESULTS: SP positive varicosities were observed in spinal lamina II. Compared with control group, the number of SP positive varicosities on the injured side apparently increased 3 days after hSCI, but decreased 7 days after operation, then recovered to normal by 21 days after operation. Noteworthily, the number of SP positive varicosities on the intact side was higher than that on the injured side. CONCLUSION: SP, as a neurotransmitter of nociceptive information, may be related to the process of hSCI.

[Expression of Bax, Bcl-2 and caspase-3 in spared dorsal root ganglion after partial dorsal root rhizotomy].

OBJECTIVE: Investigating the expression of Bax, Bcl-2 and Caspase-3 in dorsal root ganglion after partial dorsal root rhizotomy in spared root. METHODS: Adult cats were used. The study comprised a normal control group (n=5) and three expriment groups (n=5 per group). The unilateral root rhizotomy was performed on the 15 cats of the 3 experiment groups. On the 3rd, 7th and 14th days after partial dorsal rhizotomy, the DRG (L6) of 5 cats per batch were taken and made into frozen sections and were stained under the same condition using specific Bax, Bcl-2 and Caspase-3 antibody by ABC method. The Bax, Bcl-2 and Caspase-3 positive large-, medium- and small-sized neurons in DRG were counted in the 3 experiment groups and in the normal control group. RESULTS: In the intact animals of the normal control group, the Bax, Bcl-2 and Caspase-3 immunoreactants were mainly distributed in medium, small neurons and a few large neurons. The number of Bax positive neurons in spared DRG of the 3rd day group increased apparently as compared with that of the normal control group but decreased apparently in the 14th day group and showed no significant difference when compared versus control (P>0.05). There was no significant difference between the 14th day group and normal control group. Bcl-2 expression in spared DRG displayed no difference among the four groups. The number of Caspase-3 positive neurons in spared DRG of the 3rd day group increased apparently when compared against control (P<0.05), but it was not significantly different from that of the 7th day and 14th day groups (P>0.05). CONCLUSION: Partial dorsal root rhizotomy mainly has an effect on the expressions of Bax, Bcl-2 and Caspase-3 in medium- and small-sized neurons of spared DRG, the peaks of the expressions may appear 3-7 d after the operation.