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Objective:To explore the modulatory effects of GSK-3β/β-catenin signaling pathway on anxiety- and depression-like behavior of mice induced by chronic sleep deprivation (CSD).Methods:Forty-eight 10-week-old C57BL/6J male mice were selected and randomly divided into four groups(with 12 mice in each group): control group, inhibitor-only group (LiCl), chronic sleep deprivation group (CSD) and inhibitor with CSD group (LiCl+ CSD). Elevated plus maze (EPM) and forced swimming test (FST) were used to evaluate the behavior of mice, HE staining was used to observe the pathological morphological changes of hippocampal neurons, and Western blot was used to detect the protein expressions of β-catenin, GSK-3β and p-GSK-3β in hippocampal tissues. SPSS 22.0 software was used for independent sample t-test and one way ANOVA. Results:After modeling, the body weight of mice in the CSD group ((26.53±0.76)g) was significantly lower than that of the control group ((28.00±0.37)g) ( q=4.119, P=0.041), and the body weight in the LiCl+ CSD group ((28.04±0.86)g) was improved compared with CSD group ( q=4.240, P=0.036). In EPM, the ratio of the entering times and the proportion of the staying time in the open arm in the CSD group ((48.44±9.16)%) and ((16.47±10.42)%) were significantly lower than those in the control group ((68.92±11.71)% and (42.93±15.89)%) ( q=4.660, P=0.018, q=4.346, P=0.029), but the staying time in the open arm in the LiCl+ CSD group ((32.92±12.05)%) was significantly higher than that in the CSD group ( q=2.432, P=0.038). In FST, the percentage of floating immobility time of the mice in the CSD group ((55.00±5.36)%) was significantly longer than that of the control group ((39.95±2.87)%) ( P=0.023), which was decreased significantly in the LiCl+ CSD group ((42.00±7.92)%) than that in the CSD group ( P=0.040). Western blot results showed that, the expressions of p-GSK-3β and β-catenin in the hippocampus of CSD group were decreased significantly ( P=0.040, P=0.008), while the expression of GSK-3β was significantly increased than that of the control group ( P<0.001). Both p-GSK-3β and β-catenin were significantly reversed in CSD+ LiCl group than that in CSD ( P=0.034, P=0.038). Conclusion:The GSK-3β/β-catenin signaling pathway may be involved in the regulation of CSD-induced anxiety and depression-like behaviors in mice.
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Objective:To investigate the effect of lithium chloride (LiCl) on the gap junctional intercellular communication (GJIC) in human Tenon capsule fibroblasts (HTFs) and its underlying mechanism.Methods:The Tenon capsule tissue of a patient who underwent strabismus surgery in Dezhou People's Hospital in April 2019 was collected and cut into tissue blocks of dimensions 1 mm×1 mm×1 mm.Primary culture and subculture were carried out, and the 4th-generation HTFs were taken for experiment.HTFs were divided into the control group and LiCl treatment group and were cultured with cell medium without or with 80 mmol/L LiCl for another 48 hours according to grouping.The cell scratch and dye labeling technique were used to label the coupling index and evaluate the GJIC function.The expression and localization of Cx43 in HTFs were detected by immunofluorescence staining.The expression levels of Cx43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The study protocol was approved by an Ethics Committee of Dezhou People's Hospital (No.2019-023). Written informed consent was obtained from the subject.Results:The cultured spindle-shaped HTFs grew adhering to the wall showing radial monolayer or vortexlike, and the cytoplasm was vimentin positive.Results of dye tracer experiment of cell scratch showed that the cell coupling index of LiCl treatment group was 9.04±0.53, which was significantly higher than 4.94±0.39 of the control group ( t=-18.79, P<0.01). Immunofluorescence staining showed that the Cx43 fluorescence was dotted in the cell membrane between adjacent cells in the control group, and Cx43 staining was obviously enhanced in the LiCl treatment group.The results of real-time fluorescence quantitative PCR showed that with relative expression level of Cx43 mRNA in the control group set to 1, the relative expression level of Cx43 in the LiCl treatment group was significantly increased to 1.97±0.23, showing a statistical significance between them ( t=-14.426, P<0.01). Western blot showed that the relative expression level of Cx43 protein was 0.871±0.057 in the LiCl treatment group, which was significantly higher than 0.446±0.028 in the control group ( t=-11.682, P<0.01). Conclusions:LiCl can enhance the GJIC function between HTFs by upregulating the expression levels of Cx43 mRNA and protein, suggesting that the enhanced GJIC function by LiCl may be one of the mechanisms of its inhibition on HTFs proliferation.
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Abstract The purpose of this investigation was to evaluate the effects of lithium chloride (LiCl) on the socket healing of estrogen-deficient rats. Seventy-two rats were allocated into one of the following groups: Control, Ovariectomy and LiCl (150 mg/kg/2 every other day orally) + Ovariectomy. Animals received LiCl or water from the 14th day post-ovariectomy, until the completion of the experiment. On the 21st day after ovariectomy, the first molars were extracted. Rats were euthanized on the 10th, 20th and 30th days following extractions. Bone healing (BH), TRAP positive cells and immunohistochemical staining for OPG, RANKL, BSP, OPN and OCN were evaluated. The Ovariectomy group presented decreased BH compared to the LiCl group at 10 days, and the lowest BH at 20 days (p<0.05). At 30 days, the Ovariectomy and LiCl-groups presented lower BH than that of the Control (p<0.05). The number of TRAP-stained cells was the lowest in the LiCl group at 20 days and the highest in the Ovariectomy group at 30 days (p<0.05). At 10 days of healing, the LiCl group demonstrated stronger staining for all bone markers when compared to the other groups, while the Ovariectomy group presented higher RANKL expression than that of the Control (p<0.05). LiCl enhanced bone healing in rats with estrogen deficiency, particularly in the initial healing phases. However, as data on the effects of lithium chloride on bone tissue are still preliminary, more studies related to its toxicity and protocol of administration are necessary before its application in clinical practice.
Resumo O objetivo deste estudo foi avaliar os efeitos do Cloreto de Lítio (ClLi) na cicatrização de alvéolos de ratas deficientes em estrogênio. Setenta e duas ratas foram alocadas em um dos seguintes grupos: Controle, Ovariectomia e Cloreto de Lítio (150mg/kg/ oralmente a cada 2 dias) + ovarectomia. Os animais receberam ClLi ou água a partir do 14º dia pós-ovariectomia, até a conclusão do experimento. No 21º dia após a ovariectomia, os primeiros molares foram extraídos. As ratas foram sacrificadas nos dias 10, 20 e 30 após extrações. Foram avaliadas a cicatrização óssea (BH), células positivas para TRAP e coloração imuno-histoquímica para OPG, RANKL, BSP, OPN e OCN. O grupo Ovariectomia apresentou BH diminuída em comparação ao grupo LiCl aos 10 dias e a menor BH aos 20 dias (p<0,05). Aos 30 dias, os grupos Ovariectomia e LiCl apresentaram menor BH do que o Controle (p<0,05). O número de células positivas para TRAP foi menor no grupo ClLi em 20 dias e o maior no grupo Ovariectomia em 30 dias (p<0,05). Aos 10 dias de cicatrização, o grupo ClLi demonstrou imunomarcação mais intensa em todos os marcadores testados quando comparado aos outros grupos, enquanto o grupo Ovariectomia apresentou maior expressão de RANKL do que a do controle (p<0,05). O ClLi melhorou a cicatrização óssea em ratos com deficiência de estrogênio, particularmente nas fases iniciais do reparo. No entanto, como os dados sobre os efeitos do cloreto de lítio no tecido ósseo ainda são preliminares, mais estudos relacionados à sua toxicidade e protocolo de administração são necessários antes de sua aplicação na prática clínica.
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Humans , Animals , Female , Rats , Tooth Extraction , Lithium Chloride , Wound Healing , Ovariectomy , Rats, Wistar , Tooth Socket , EstrogensABSTRACT
RESUMO No presente trabalho, teve-se por objetivo avaliar a influência da proporção geométrica e o tempo de operação na hidrodinâmica do sistema. Para isso, três sistemas alagados construídos de escoamento horizontal subsuperficial (SACs-EHSS) de diferentes razões comprimento:largura (L/B) foram caracterizados no que se refere ao comportamento hidrodinâmico, no início e após três meses de sua operação, durante o tratamento de esgoto sanitário. O comportamento hidrodinâmico foi avaliado por meio da técnica estímulo-resposta, com a injeção de traçador de cloreto de lítio (LiCl), na forma de pulso, e da construção da curva de distribuição dos tempos de residência (DTR), a partir da qual foram extraídos parâmetros que permitiram a análise hidrodinâmica dos sistemas. Verificou-se que todos os SACs-EHSS apresentaram comportamento hidrodinâmico do tipo disperso, embora tenha sido observada correlação inversa entre L/B e o número de dispersão (d) no sistema. O tempo de operação desses sistemas também proporcionou pequena redução no d, em todos os SACs-EHSS.
ABSTRACT The objective of the present work was to evaluate the geometric ratio and the operating time's influence on the systems' hydrodynamic conditions. For this purpose, three horizontal subsurface flow constructed wetlands (HSSF-CWs) in pilot scale, with same useful volume () but with different length:width (L/B) ratio, were characterized regarding the hydrodynamic behavior at the beginning and after 3 months of operation, in sewage treatment. To obtain the HSSF-CW conditions, the stimulus-response technique with the lithium chloride (LiCl) tracer injection on a pulse format was used. Thus, the residence time distribution curve was constructed, from which parameters were extracted and enabled the hydrodynamic analysis of the systems. It was found that all HSSF-CWs presented dispersed hydrodynamic behavior, although the results indicated an inverse correlation between L/B and the dispersion number. The operating time of these systems provided a reduction in the dispersion number in all HSSF-CWs, different from the results found in some works.
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@#AIM: To study the expressions of TGFBI and microtubule-associated protein 1 light chain 3 alpha(LC3)in granular corneal dystrophy, and the influences of lithium chloride(LiCl)on corneal stromal fibroblast cell proliferation by TGFBI. <p>METHODS: Immunohistochemistry and Western-blot assays were used to detect the expression levels of TGFBI and LC3 in corneal dystrophy and normal corneal tissues. TGFBI overexpression vector was transfected into corneal stromal fibroblasts, and then the cells were treated with 5, 10, 20, 40mmol/L LiCl for different times(0, 1, 6, 12h), and Western-blot assay was performed to evaluate the expression levels of TGFBI and LC3, and CCK-8 assay was carried out to assess cell proliferation activity.<p>RESULTS: TGFBI and LC3 were highly expressed in corneal tissues of patients with corneal dystrophy. TGFBI overexpression inhibited the proliferation ability of corneal stromal fibroblasts(<i>P</i><0.05). LiCl inhibited the expression levels of TGFBI and LC3, and enhanced the cell proliferation activity in corneal stromal fibroblasts transfected with mutant TGFBI(<i>P</i><0.05).<p>CONCLUSION: LiCl promoted the proliferation and autophagy of corneal stromal fibroblasts, and its mechanism may be related to down regulated expressions of TGFBI and LC3.
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Objective@#To investigate the effect and significance of GSK-3β inhibitor(LiCl)and RANK-RANKL on the renal tissue of diabetic nephropathy(DN) rats.@*Methods@#SD rats were divided into normal control group (NC), DN model group (DN) and GSK-3β inhibitor intervention group (LiCl). Twenty-four hour urine protein of rats were determined by Coomassie brilliant blue. Kidney tissue sections were stained by HE. The expression of GSK-3β, RANK and RANKL protein were determined by immunohistochemistry staining. The mRNA of GSK-3β, RANK, RANKL was detected by RT-qPCR.@*Results@#Compared with NC group[(14.72±3.37)g], the level of 24-hour urinary protein[(154.17±20.65)g] increased significantly in DN group; compared with DN Group, the level of 24-hour urinary protein [(107.22±31.15)g]decreased in LiCl group(P<0.05). Compared with NC group(2.10±0.60, 1.10±0.20, 1.21±0.20; 19.52±3.20, 1.80±1.10, 1.81±0.50), the pathological changes of renal tissues of DN group aggravated, the mRNA and expression of protein of GSK-3β, RANK and RANKL increased(9.10±2.15, 8.95±2.40, 9.90±2.60; 32.70±7.20, 19.20±4.32, 20.92±5.90); compared with DN group, the pathological changes of renal tissues of LiCl group alleviated, mRNA and the expression of protein of factors above declined(2.70±0.80, 2.32±0.65, 3.58±1.10; 22.35±3.25, 4.20±2.42, 5.90±2.36; P<0.05).@*Conclusion@#RANK and RANKL play an important role in the development of DN, LiCl influence Wnt and NF-κB signal pathway down-regulating RANK and RANKL to suspend development of diabetic nephropathy.
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Objective To study the effect of lithium chloride on the gap junction in the myocardium under chronic hypoxia.Methods Twenty-five C57BL/6J mice were randomly divided into normoxia group,hypoxia group,normoxic control group,hypoxia + saline group and hypoxia + lithium chloride group.Hypoxia group was treated with 10％ oxygen concentration for 4 weeks.Hypoxia + saline group and hypoxia + lithium chloride group were intraperitoneal injection of saline and lithium chloride.Electrophysiology and cardiac catheterization were used to assess arrhythmias,heart rate and ejection fraction.The expression of Cx43,phosphorylated glycogen synthase kinase 3β(p-GSK-3β) and glycogen synthase kinase 3β (GSK-3β) were detected by Western blot.Results Compared with the normoxia group,the hypoxia group had a faster heart rate [(448 ± 18) bpm vs.(401 ± 13) bpm,P＜0.05),and the ejection fraction was decreased [(56±5)％ vs.73±4)％,P＜0.05],arrhythmia score increased [(3.4±0.5)％ vs.(0.6±0.5)％,P＜0.05],Cx43 expression was decreased.Compared to hypoxia + normal saline group,the heart rate decreased[(412±11)bpm vs.(454±18)bpm,P＜0.05],ejection fraction increased[(69±3)％ vs.(55±4)％,P＜0.05],the score of arrhythmia decreased [(1.8±0.4) ％ vs.(3.0±0.7)％,P＜0.05] in hypoxia + lithium chloride group,the expression of Cx43 and the rate of p-GSK-3β to GSK-3β were increased.Conclusion During the chronic hypoxia,lithium chloride can sustain the gap junction through inhibition of GSK-3β signaling way,which can also reduce the rate of arrhythmia.
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AIM:To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process .METHODS:MSCs were isolated and cultured in vitro.The cells were divided into LiCl group and control group .MSCs were treated withβ-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons .The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associ-ated protein 1 light chain 3 ( LC3) were measured by immunofluorescence method and Western blot .An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells.The protein expression of NSE and MAP-2 were determined by Western blot .RESULTS: After induction, the ex-pression of NSE and MAP-2 were increased .The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05).After induction, the number of LC3-positive dots and the expression of LC3-Ⅱin LiCl group were greater than those in control group (P<0.05).The expres-sion of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated -cells, and on the contra-ry, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA.CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy .
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Objective To investigate the effects of different interventions on the biomechanic indexes in femur bones of ovariectomized rats with osteoporosis.Methods Eighty healthy female Sprague-Dawley rats,aged 3 months,were randomly divided into an ovariectornized(OVX,n=68)group and a sham-operation(Sham,n=12)group.At the 11th week after the operation,the OVX group was further randomly divided into a control group(n=11),an estradiol group(E2,n=10),a genisteine group(G,n=10),a treadmill exercise group(TE,n=10),a lithium chloride group(LiCl,n=10)and a whole-body vertical vibration group (WBVV,n=10).Then the rats began to receive different interventions as their group names implied.At the end of 8 weeks,their right femurs were isolated and the biomechanic parameters were detected using the three point bending test.Results (1)Both the maximum load and elastic load in E2,G,TE,WBVV and LiC1 groups were significantly higher than that of the control group,while no significant differences were seen in maximum deflection and elastic deflection.(2)The elastic stress,maximum strain,and elasticity modulus in E2,G,and WBVV groups were significantly higher than that of the control group,while no significant differences were seen in maximum stress and elastic strain among them.The maximum stress,elastic stress,maximum strain,and elasticity modulus in TE group were significantly higher than the control group while no significant differences were seen in the elastic strain.The maximum stress,elastic stress,and elasticity modulus in LiC1 group were significantly higher than the control group,but no significant differences were seen in maximum strain and elastic strain between them.Conclusion E2,genistein,treadmill exercise,whole-body vertical vibration,and LiC1 have quite similar effects on structural mechanics indexes of femurs in OVX rats with osteoporosis,but they have different impacts on the material mechanics indexes.
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AIM:To research the effects of lithium chloride on transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) in cultured human Tenon capsule fibroblasts (HTFs) and explore its mechanism.METHODS:HTFs were cultured and identified by vimentin staining with immunofluorescence and the morphological characteristics.The experimental group was processed 48h with LiCl in concentration of 80mmol/L, the control group without LiCl.The mRNA expression of TGF-β and CTGF in two groups were analyzed with real-time fluorescent quantitative polymerase chain reaction (real time-qPCR) and the protein expression was detected with Western blot.RESULTS:The cultured HTFs expressed TGF-β and CTGF.The mRNA expression of TGF-β and CTGF significantly decreased compared with the control group(t=20.042, 14.995, P<0.05).the protein expression of TGF-β and CTGF also decreased significantly compared with the control group(t=46.058、12.452, P<0.05)CONCLUSION:The cultured HTFs can express TGF-β and CTGF in mRNA and proteins' level.LiCl can reduce the expression of TGF-β and CTGF both in gene and proteins' level.LiCl has the potential to modulate wound healing for glaucoma filtration surgery.
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Objective To investigate the protective effect of lithium chloride (LiCl)-activated Wnt/beta-catenin signaling pathway on glucocorticoid-induced bone loss. Methods C57BL6/J mice were randomly divided into 3 groups. osteoporosis (OP) group and LiCl group were injected intraperitoneally once daily with dexamethasone (50 mg kg). while control group was injected with normal saline. LiCl group was given LiCl (200 mg kg) through water administration. After 5 weeks of treatment, mice were sacrificed within 24 h. Anti-active hela-catenin antibody was used in immuno his to chemical staining to detect the activity of Wnt/beta-cat enin signaling pathway, tone morphology was observed by UK siaining. Meanwhile. bone histomorphometrical parameters were analyzed by microCT. Bone formation and bone resorption of different groups were detected by calcein labeling and TRAP siaining. respectively. Results The protein level of active beta-calenin was higher in LiCl group compared with OP group, tone histomorphometrical parameters including the trabecular bone volume as a percentage of total volume (BV/TV). bone mineral density (HMD), trabecular number (Tb. N) and trabecular space (Tb. Sp) were all significantly improved in LiCl group- even though obvious difference existed lieiween LiCl group and control group (P<0. 05). Calcein labeling showed that new hone formation was markedly improved in LiCl group, but was still lower than that in control group, tone resorption was not significantly changed among three groups as indicated by TRAP siaining. Conclusion LiCl administration effectively activates Wnt/β-catenin signaling pathway in vivo* which improve new bone formation and rescue glucocorticoid-induced bone loss.
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Objective To evaluate the role of Wnt/β-catenin signaling pathway in lithium chloride preconditioning-induced improvement in postoperative cognitive function of aged rats.Methods A total of 100 pathogen-free healthy male Sprague-Dawley rats,aged 18 months,weighing 540-650 g,were divided into 5 groups (n =20 each) using a random number table:control group (group C),surgery group (group S),lithium chloride preconditioning group (group L),secreted frizzled-related protein 1 (sFRP-1) group (group F) and lithium chloride preconditioning plus sFRP-1 group (group L+F).Lithium chloride 2 mmol/kg was intraperitoneally injected once a day for 5 consecutive days before operation in L and L+ F groups.In F and L + F groups,sFRP-1 10 μl (concentration 10 μg/ml) was injected into the ventricle at 1 day before operation.Ten rats in each group were randomly sacrificed at 1 day after operation,and the hippocampi were removed for determination of the expression of phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β),3-catenin and Wnt in hippocampal tissues (by Western blot).The rest rats underwent Morris water maze test at day 3-7 after operation,and the concentrations of amyloid beta 42 (A3-42) and phosphorylated tau-181 protein (p-tau-181) in cerebrospinal fluid (CSF) were detected by enzyme-linked immunosorbent assay at day 7 after operation.Results Compared with group C,the escape latency was significantly prolonged at 3-7 days after operation,and the concentration of Aβ-42 in CSF was increased in the other four groups,and the expression of Wnt,p-GSK-3β and β-catenin in hippocampal tissues was down-regulated in group S,the expression of Wnt,p-GSK-3β and β-catenin in hippocampal tissues was significantly up-regulated in L and L+F groups,and the concentration of p-tau-181 in CSF was significantly increased in S and L+F groups (P＜0.05).Compared with group S,the escape latency was significantly shortened at 3-7 days after operation in group L and at 5-7 days after operation in group L+F,and the expression of Wnt,p-GSK-3β and β-catenin was significantly up-regulated,and the concentrations of Aβ-42 and p-tau-181 in CSF were decreased in L and L+F groups (P＜0.05).Compared with group L,the escape latency was significantly prolonged at 3-6 days after operation,the expression of Wnt,p-GSK-3β and β-catenin was down-regulated,and the concentration of p-tau-181 in CSF was increased in group L+F (P＜ 0.05).Compared with group F,the escape latency was significantly shortened at 3-7 days after operation,the expression of Wnt,p-GSK-3β and β-catenin was up-regulated,and the concentrations of Aβ-42 and ptau-181 in CSF were decreased in group L+F (P＜0.05).Conclusion The mechanism by which lithium chloride preconditioning improves in postoperative cognitive function is partially related to activating Wnt/β-catenin signaling pathway in aged rats.
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ABSTRACT: The objective of this study was to describe the epidemiological, clinical, and pathological aspects of Palicourea aeneofusca poisoning in cattle in the region of Pernambuco, Brazil and to determine if it is possible to induce food aversion by P. aeneofusca poisoning in cattle raised under extensive management conditions. To determine the occurrence of poisoning, 30 properties were visited in five municipalities of the region of Pernambuco. Three outbreaks of poisoning of cattle were monitored. To induce conditioned food aversion by the consumption of P. aeneofusca, 12 animals were randomly distributed into two groups of six animals each. Cattle were weighed and received green P. aeneofusca leaves in their trough at a dose of 35mg kg-1 body weight for spontaneous consumption. The control group (CG) animals received water (1ml kg-1 body weight) via a feeding tube after the first ingestion of the plant, while the other animals, constituting the aversion test group (ATG), underwent induced aversion with lithium chloride (LiCl - 175mg kg-1 body weight) via a feeding tube. For the ATG cattle, the aversion to P. aeneofusca induced by a single dose of LiCl persisted for 12 months. In contrast, the CG animals continued to consume the plant in all tests performed, indicating the absence of aversion. This study showed that aversive conditioning using LiCl was effective in preventing poisoning by P. aeneofusca for a period of at least 12 months.
RESUMO: O objetivo deste trabalho foi descrever os aspectos epidemiológicos, clínicos e patológicos da intoxicação por Palicourea aeneofusca em bovinos, no Agreste de Pernambuco, e comprovar se é possível induzir aversão alimentar à intoxicação por P. aeneofusca em bovinos criados sob manejo extensivo. Para determinar a ocorrência da intoxicação, foram visitadas 30 propriedades em cinco municípios do Agreste de Pernambuco. Três surtos de intoxicação em bovinos foram acompanhados. Para se induzir aversão alimentar condicionada ao consumo de P. aeneofusca, 12 bovinos foram distribuídos aleatoriamente em dois grupos de seis animais cada. Os bovinos foram pesados e receberam, no cocho, folhas verdes de P. aeneofusca, na dose de 35mg kg-1 de peso corporal, para consumo espontâneo. Os bovinos do GC receberam água (1mL kg-1 de peso corporal), via sonda esofágica, após a primeira ingestão da planta, e os demais constituíram o GTA, que foram induzidos à aversão com cloreto de lítio (LiCl - 175mg kg-1 de peso corporal), via sonda esofágica. Para os bovinos do GTA, a indução de aversão a P. aeneofusca, em que se utilizou dose única de LiCl, persistiu por 12 meses. Por outro lado, os bovinos do grupo GC continuaram ingerindo a planta em todos os testes realizados, indicando a ausência de aversão. Este trabalho comprova que o condicionamento aversivo usando LiCl foi eficaz para prevenir a intoxicações por P. aeneofusca por um período de, pelo menos, 12 meses.
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OBJECTIVE: To investigate the effects of lithium chloride (LiCl) on neuroapoptosis in hippocampus induced by isoflurane and the relationships with GSK-3β/CRMP2 pathway in neonatal rats. METHODS: Sixty neonatal rats at postnatal day 7 were assigned randomly into air + NS group, air + LiCl group, Iso + NS group, Iso + LiCl2, Iso + LiCl4 and Iso + LiCl8 group. Rats in groups of air + NS and air + LiCl were exposed to air for 6 h. Rats in groups of Iso + NS, Iso + LiCl2, Iso + LiCl4 and Iso + LiCl8 were exposed to 0.75% isoflurane for 6 h. Rats in groups of Air + NS and Iso + NS were injectedintracerebroventricularlywith NS 5 μL separately 30 min before exposure to air or isoflurane. Rats in groups of air + LiCl, Iso + LiCl2, Iso + LiCl4 and Iso + LiCl8 were injected intracerebroventricularly with LiCl 5 μL separately 30 min before exposureto air or isoflurane. Rats in groups of Iso + LiCl2, Iso + LiCl4, Iso + LiCl8 and Air + LiCl were injected intracerebroventricularly with LiCl5 μL separately 30 min before exposure with the different concentrations(20, 40, 80, 80 mmol·L-1, respectively). At the end of exposure, the hippocampus of some rats were separated and proteins expression of cleaved caspase-3, phospho-GSK-3β(p-GSK-3β), GSK-3β, phospho-CRMP2 (p-CRMP2) and CRMP2 were detected by Western blot (n=5); other rats were perfused and their brain were embedded by paraffin 2 h after exposure, the neuroapoptosis in the hippocampus CA1 area was detected by TUNEL staining (n=5). RESULTS: Lithium chloride (80 mmol·L-1) significantly decreased the cleaved caspase-3 levels(P < 0.01) induced by isoflurane in hippocampus of neonatal rats. TUNEL positive cells in CA1 area were significantly decreased by 41.69%(P < 0.01) in group Iso + LiCl8 when compared with those in group Iso + NS. The protein expressionof p-GSK-3β and p-CRMP2 in group Iso + NS were significantly decreased and increased respectivelycompared to those in group air + NS. The ratio of p-GSK-3β/GSK-3β and p-CRMP2/CRMP2 in the hippocampal increased 41.03% (P < 0.05) and decreased 23.64%(P < 0.05) respectively in Iso + LiCl8 group when compared with Iso + NS group. CONCLUSION: Lithium chloride attenuates isoflurane-induced neurodegeneration through inhibiting hippocampal neuroapoptosis in neonatal rats. Inhibition of the activation of GSK-3β/CRMP2 signaling pathways may involve in the mechanisms.
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A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 µl of Murashige and Skoog’s medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 106 cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.
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BACKGROUND:Osteoporosis is a bone metabolic disease that affects women more than men. Prevention and treatment of osteoporosis is becoming a serious medical problem because of the aging of the population. OBJECTIVE:To explore the effects of lithium chloride treatment on bone microarchitecture and bone marrow stromal cel differentiation of ovariectomized osteoporosis rats. METHODS:After ovariectomy, 28 of 30 healthy female Sprague-Dawley rats, 3 months old, were randomly divided into the folowing three groups: ovariectomizedin vivo group (9 rats), ovariectomizedin vitro group (10 rats), and lithium chloride group (9 rats). At the 11th week postoperatively, rats in the lithium chloride were intragastricaly injected with lithium chloride at a dose of 15 mg/kg, three times per week. After 8 weeks of treatment, the bone microarchitectures of the rat left femur in the ovariectomizedin vivo group and lithium chloride group were detected by micro-CT. The bone marrow mesenchymal stem cels were freshly isolated from the bone marrow of the bilateral femurs and tibia of rats in the ovariectomizedin vitro group. After 24 hours of inoculation, the cels were cultured in lithium chloride and divided into 0 mmol/L (control), 1 mmol/L and 5 mmol/L groups. At 6 and 8 days of culture, the medium was changed and lithium chloride with the corresponding concentrations was added. At 10 days of culture, western blot assay was adopted to detect protein expression of Runx-2, SP7 and PPARγ2. RESULTS AND CONCLUSION:(1) Compared with the ovariectomizedin vivo group, the volume density of trabecular bone, number of trabecular bone, and bone volume fraction in the lithium chloride group were significantly increased and the separation of trabecular bone was significantly decreased. However, no differences were seen in the thickness of trabecular bone and structure model index. (2) Lithium chloride at 1 and 5 mmol/L could increase the protein expression of Sp7 and Runx-2 in bone marrow stromal cels, but decrease the protein expression ofPPARγ2. These results indicate that lithium chloride may improve the microarchitecture of the trabeculr bone in ovariectomized osteoporosis rats through stimulating the osteogenic differentiation of bone marrow stromal cels.
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Ipomoea carnea is a toxic plant often ingested by livestock in Brazil. Three experiments were conducted to determine if conditioned food aversion was effective in reducing goats' consumption of I. carnea. In the first experiment, 10 mildly intoxicated goats that had been eating I. carnea were averted using LiCl (175 to 200mg kg-1 body weight). These intoxicated goats did not develop an aversion to I. carnea, demonstrating that the technique is not effective in goats that are already accustomed to consuming the plant. In the second experiment, 14 naïve goats were placed in a pasture with I. carnea, and averted after they ingested the plant. In this group the aversion persisted until the end of the experiment, 2 years and 8 months after the initial aversion. In another experiment, 20 goats were placed in a pasture with I. carnea, and after consuming the plant were averted with LiCl. The averted goats were transferred to Marajo Island and periodically observed over a 2 year period at 2-3 month intervals to determine if they were still averted. The averted goats did not ingest the plant while grazing in the pasture, whereas in 6 neighboring goat farms the prevalence of intoxication from I. carnea poisoning was estimated to be about 40%. These results demonstrated the efficacy of conditioned food aversion to avoid ingestion of I. carnea in formerly naïve goats that had only recently begun to ingest the plant.
Para testar a técnica de aversão alimentar condicionada como método de controle para a intoxicação por I. carnea, foram realizados 3 experimentos administrando cloreto de lítio (LiCl) na dose de 175-200mg kg-1 após a ingestão da planta por caprinos. No primeiro, foram induzidos à aversão 10 caprinos que tinham o hábito de ingerir a planta e com sinais clínicos da intoxicação. Apesar da realização de diversos tratamentos aversivos, após os animais ingerirem a planta, a aversão não foi eficiente, demonstrando que a técnica não é eficiente em caprinos que já estão habituados a ingerir a planta. No segundo experimento, 14 caprinos foram adaptados a ingerir a planta na pastagem e, após ingerirem a planta a campo, foram induzidos à aversão com LiCl. Neste grupo, a aversão persistiu até o fim do Experimento, 2 anos e 8 meses após a aversão. Em outro experimento, 20 caprinos foram adaptados a consumir I. carnea e, em seguida, induzidos à aversão com LiCl. Esses animais foram transferidos para uma propriedade na Ilha de Marajó, onde foram realizadas 9 visitas com intervalos de 2-3 meses para verificar a duração da aversão. Após 2 anos de observações, nenhum animal voltou a ingerir a planta na pastagem e não foram observados casos de intoxicação, enquanto que, em 6 propriedades vizinhas, a doença foi observada com uma prevalência de até 60%. Esses resultados demonstram a eficiência da aversão alimentar condicionada para evitar a ingestão de I. carnea em caprinos recém adaptados a ingerir a planta, nas regiões invadidas por esta planta e nas condições naturais da Ilha de Marajó.
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Objective To explore the effect of liensinine on cortical EEG of epileptic rats induced by lithium chloride-pilocarpine,and investigate the effective spectrum of liensinine on epilepsy.Methods 32 SD rats were randomly divided into four groups:low dose of liensinine group(2.5 mg/mL, 10μL),high dose of liensinine group(5 mg/mL,10μL),the normal saline group(10μL)which was negative control group,levetiracetam group (100 mg/mL,10μL)which was positive control group,8 rats in each group.Electrocorticogram of rats was recorded after chloride lithium-pilocarpine model was induced.The anesthetic rats were fixed on stereotaxic apparatus after the epilepsy model was confirmed by ethology.A trochar was put into the left lateral ventricle.Rats were implanted with epidural recording electrodes.After the cortical EEG was recorded about 30 minutes, liensinine (at concentration of 2.5,5 mg/mL),levetiracetam and 0.9% sodium chloride was injected into lateral ventricle.Electrocorticogram was recorded about 150 minutes again.The frequency of epileptic discharge was observed every 30 minutes.The differences of frequency in the same group and the different change of frequency between groups at the same period were compared.Results The frequency of epileptic discharge decreased in low dose of liensinine group,high dose of liensinine group and levetiracetam group after administration ,there was significantly statistical difference in low dose of liensinine group after administration about 60 minutes(P<0.01 ),there was significant statistics difference in high dose of liensinine group after administration about 30 minutes(P<0.01),and the same change in levetiracetam group within 30 minutes after administration(P<0.01);the change of frequency of epileptic discharge was no significantly statistical difference between pro-and post-administration in the normal saline control group.The difference of the frequency change in epileptic discharge at the same period between liensinine group and levetiracetam group was observed ,there was statistic difference between low dose of liensinine group and levetiracetam group at the period of thirty to sixty minutes after administration,there was no statistic difference at other periods;there was no statistic difference between high dose of liensinine group and levetiracetam group at every period.Conclusion Liensinine could inhibit the epileptic discharges in acute model of epileptic rats induced by chloride lithium-pilocarpine.
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BACKGROUND:The proliferation and differentiation of hair-folicle-generating stem cels are influenced by the joint action of their own genes and external signals. Wnt/β-catenin signaling pathway plays an important role in the development of hair folicles, but the detailed mechanisms are not yet clear. OBJECTIVE:To investigate, with interruption of keratinocyte growth factor and lithium chloride, the function and the interrelationship of Wnt/β-catenin signaling pathway with other signal factors when human hair-folicle-generating stem cels differentiate into dermal papila cels or epidermal cels. METHODS: Hair-folicle-generating stem cels were isolated from the bulge and cultivated. Then the growth curve of hair-folicle-generating stem cels was tested and formed in order to observe the cellproliferation ability cultivated at different densities (1×106/L, 1×107/L, 1×108/L, 1×109/L) at each time. Immunoflurorescene staining was performed to identify hair-folicle-generating stem cels and their differentiated cels. Lithium chloride (0, 0.5, 1.5, 10, 25 mmol/L individualy) and keratinocyte growth factor(0, 10, 25, 50, 100 μg/L individualy) were used to induce the differentiation of hair-folicle-generating stem cels. Then, we contrasted and analyzed the proliferation ability in each case, thereby investigating the most appropriate concentration of keratinocyte growth factor and lithium chloride to spur the differentiation of hair-folicle-generating stem cels. At days 3, 5, 7 and 9, we tested and compared the mRNA expressions of β-catenin, APC, GSK-3β, Axin and Lef1 from cels in control group, 10 mmol/L lithium chloride group and 10 μg/L keratinocyte growth factor group. RESULTS AND CONCLUSION:Isolating cultured hair-folicle-generating stem cels stil had a great reproductive activity and multi-lineage potential even after various times subculturein vitro. With higher lithium chloride concentration, the proliferation ability of hair-folicle-generating stem cels declined; while it increased when keratinocyte growth factor concentration increased. In K-SFM medium which contained lithium chloride, the transformation of hair-folicle-generating stem cels was obvious, showing distinct differences among groups. Especialy, the level ofβ-catenin reached the peak when lithium chloride > 10 mmol/L. However, in K-SFM medium which contained keratinocyte growth factor, hair-folicle-generating stem cels differentiated into epidermal cels and the level of β-catenin changed slightly. We found that, while spurring the differentiation of hair-folicle-generating stem cels, lithium chloride could activate Wnt/β-catenin signal pathway and inhibit GSK-3β, a vital component of degradation compound. This facilitated β-catenin expressing in the cytoplasm to translocate into the nucleus. As a result, the transcription of target gene increased. It is the most appropriate concentration to spur hair-folicle-generating stem cels differentiation when lithium chloride level is > 10 mmol/L, but the proliferation ability declines correspondingly. Keratinocyte growth factor, which can facilitate hair-folicle-generating stem cels differentiated into epidermal cels, is a key factor to accelerate proliferation ability and migration of hair-folicle-generating stem cels, re-epithelialization and healing of wound. The mechanisms of hair-folicle-generating stem cels oriented differentiation induced by lithium chloride and keratinocyte growth factor are activating Wnt/β-catenin signal pathway, inducing change of β-catenin expression, and activating the transcription of target gene related to Wnt/β-catenin signaling pathway .
ABSTRACT
A aversão alimentar condicionada é uma técnica que pode ser utilizada em animais para evitar a ingestão de plantas tóxicas. O presente estudo teve como objetivo testar a eficiência e durabilidade da aversão alimentar condicionada em caprinos para evitar o consumo de Ipomoea carnea subsp. fistulosa. Foram utilizados 14 caprinos jovens da raça Moxotó, que foram adaptados ao consumo da planta. Inicialmente foi administrada I. carnea subsp. fistulosa dessecada e triturada misturada à ração concentrada por 30 dias e, posteriormente, foi fornecida a planta verde por mais 10 dias. Para constatação da adaptação ao consumo da planta os caprinos foram colocados a pastar em um piquete de 510 m² onde tinha sido plantada I. carnea subsp. fistulosa em uma área de 30m² (10 plantas/m²). No 42º dia de experimento, após a constatação do consumo espontâneo os animais receberam a planta verde individualmente na baia por alguns minutos, e todos os animais que consumiam qualquer quantidade da planta foram tratados com uma solução de LiCl na dose 175mg por kg de peso vivo. Este procedimento repetiu-se por mais dois dias. Posteriormente, os caprinos foram divididos em dois grupos: Grupo 1 com seis animais, quatro deles avertidos e dois não avertidos (facilitadores); e o Grupo 2, com oito caprinos, todos avertidos. Para constatar a eficiência e duração da aversão e a influência de animais facilitadores na durabilidade da aversão, os caprinos foram colocados a pastar, em dias alternados, três dias por semana, durante duas horas, no piquete plantado com I. carnea subsp. fistulosa. Por 12 meses os animais foram monitorados durante o pastejo, identificando-se o consumo e a preferência dos animais pelas plantas presentes no piquete. No Grupo 1 tanto os caprinos avertidos quanto os não avertidos iniciaram a ingerir a planta em 1-6 semanas e gradualmente foram aumentando a planta consumida, mas nunca a ingeriram exclusivamente. Nenhum caprino do Grupo 2 iniciou a ingestão da planta durante os 12 meses de experimento. Após esse período a área do piquete destinada ao plantio de I. carnea subsp. fistulosa foi ampliada para 80m² e os animais foram novamente introduzidos, com tempo de pastejo na área aumentado para quatro horas durante cinco dias na semana. Nesta fase todos os caprinos do Grupo 1 ingeriram a planta em grande quantidade. Os caprinos do Grupo 2 iniciaram gradualmente a ingerir a planta e a aversão se extinguiu, em todos os animais, após dois meses. A concentração de swainsonina em I. carnea subsp. fistulosa foi de 0,052±0,05% (média±SD). Conclui-se que a aversão alimentar condicionada é eficiente para evitar a ingestão de I. carnea subsp. fistulosa. No entanto, a duração da mesma depende, entre outras coisas, da quantidade de planta presente na área de pastoreio e do tempo de exposição e se extingue rapidamente por facilitação social.
Conditioned food aversion is a technique that can be used to train livestock to avoid ingestion of poisonous plants. This study tested the efficacy and durability of conditioned food aversion to eliminate goat's consumption of Ipomoea carnea subsp. fistulosa. We used 14 young Moxotó goats, which were initially adapted to the consumption of the plant by offering dried I. carnea subsp. fistulosa with their concentrate diet for 30 days, and then subsequently providing green plant for another 10 days. To confirm the spontaneous consumption of the plant, the goats were allow to graze in a paddock of 510m² where I. carnea subsp. fistulosa had been planted in an area of 30m² (10 plants/m²). On day 42, 12 goats were offered fresh green plant individually in a pen for a few minutes, and after the consumption of any amount of the plant they were treated orally with a solution of LiCl at a dose 175mg per kg of body weight. This procedure was repeated for two more consecutive days. Thereafter, the goats were divided into two groups: Group 1 with four averted and two non-averted goats; and Group 2 with eight averted goats. To verify the efficacy and duration of aversion, both groups were introduced into the paddock with I. carnea subsp. fistulosa three days a week for two hours daily. In Group 1, with two non-averted and four averted goats, all animals started to ingest the plant after 1-6 weeks of grazing. They continually increased their consumption of the plant, but never consumed the plant exclusively. None of the goats of Group 2 goats started eating the plant during the 12 months of observation. After this period the area of the paddock planted with I. carnea subsp. fistulosa was expanded to 80 m² and grazing time was increased to four hours per day for five days a week. At this stage all the goats in Group 1 ingested the plant in large quantities. The goats from Group 2 gradually started to eat the plant and aversion was extinguished in all animals after two months. Swainsonine concentration of I. carnea subsp. fistulosa was 0.052±0.05% (mean ±SD). It was concluded that conditioned food aversion was effective in reducing goat consumption of I. carnea subsp. fistulosa, but the duration of aversion depends on the time of grazing and amount of plant available. However, the aversion was quickly extinguished by social facilitation when averted animals grazed with non-averted animals.