Dihydrotanshinone I inhibits gallbladder cancer growth by targeting the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation.

BACKGROUND: Gallbladder cancer (GBC) poses a significant risk to human health. Its development is influenced by numerous factors, particularly the homeostasis of reactive oxygen species (ROS) within cells. This homeostasis is crucial for tumor cell survival, and abnormal regulation of ROS is associated with the occurrence and progression of many cancers. Dihydrotanshinone I (DHT I), a biologically effective ingredient isolated from Salvia miltiorrhiza, has exhibited cytotoxic properties against various tumor cells by inducing apoptosis. However, the precise molecular mechanisms by which dht I exerts its cytotoxic effects remain unclear. PURPOSE: To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms. METHODS: The proliferation of GBC cells, NOZ and SGC-996, was assessed using various assays, including CCK-8 assay, colony formation assay and EdU staining. We also examined cell apoptosis, cell cycle progression, ROS levels, and alterations in mitochondrial membrane potential to delve into the intricate molecular mechanism. Quantitative PCR (qPCR), immunofluorescence staining, and Western blotting were performed to evaluate target gene expression at both the mRNA and protein levels. The correlation between nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1) were examined using co-immunoprecipitation. Finally, the in vivo effect of dht I was investigated using a xenograft model of gallbladder cancer in mice. RESULTS: Our research findings indicated that dht I exerted cytotoxic effects on GBC cells, including inhibiting proliferation, disrupting mitochondrial membrane potential, inducing oxidative stress and apoptosis. Our in vivo studies substantiated the inhibition of dht I on tumor growth in xenograft nude mice. Mechanistically, dht I primarily targeted Nrf2 by promoting Keap1 mediated Nrf2 degradation and inhibiting protein kinase C (PKC) induced Nrf2 phosphorylation. This leads to the suppression of Nrf2 nuclear translocation and reduction of its target gene expression. Moreover, Nrf2 overexpression effectively counteracted the anti-tumor effects of dht I, while Nrf2 knockdown significantly enhanced the inhibitory effect of dht I on GBC. Meanwhile, PKC inhibitors and nuclear import inhibitors increased the sensitivity of GBC cells to dht I treatment. Conversely, Nrf2 activators, proteasome inhibitors, antioxidants and PKC activators all antagonized dht I induced apoptosis and ROS generation in NOZ and SGC-996 cells. CONCLUSION: Our findings indicated that dht I inhibited the growth of GBC cells by regulating the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation. These insights provide a strong rationale for further investigation of dht I as a potential therapeutic agent for GBC treatment.

Bavachin protects against diet-induced hepatic steatosis and obesity in mice.

Non-alcoholic fatty liver disease (NAFLD) is a major health concern worldwide, and the incidence of metabolic disorders associated with NAFLD is rapidly increasing because of the obesity epidemic. There are currently no approved drugs that prevent or treat NAFLD. Recent evidence shows that bavachin, a flavonoid isolated from the seeds and fruits of Psoralea corylifolia L., increases the transcriptional activity of PPARγ and insulin sensitivity during preadipocyte differentiation, but the effect of bavachin on glucose and lipid metabolism remains unclear. In the current study we investigated the effects of bavachin on obesity-associated NAFLD in vivo and in vitro. In mouse primary hepatocytes and Huh7 cells, treatment with bavachin (20 µM) significantly suppressed PA/OA or high glucose/high insulin-induced increases in the expression of fatty acid synthesis-related genes and the number and size of lipid droplets. Furthermore, bavachin treatment markedly elevated the phosphorylation levels of AKT and GSK-3ß, improving the insulin signaling activity in the cells. In HFD-induced obese mice, administration of bavachin (30 mg/kg, i.p. every other day for 8 weeks) efficiently attenuated the increases in body weight, liver weight, blood glucose, and liver and serum triglyceride contents. Moreover, bavachin administration significantly alleviated hepatic inflammation and ameliorated HFD-induced glucose intolerance and insulin resistance. We demonstrated that bavachin protected against HFD-induced obesity by inducing fat thermogenesis and browning subcutaneous white adipose tissue (subWAT). We revealed that bavachin repressed the expression of lipid synthesis genes in the liver of obese mice, while promoting the expression of thermogenesis, browning, and mitochondrial respiration-related genes in subWAT and brown adipose tissue (BAT) in the mice. In conclusion, bavachin attenuates hepatic steatosis and obesity by repressing de novo lipogenesis, inducing fat thermogenesis and browning subWAT, suggesting that bavachin is a potential drug for NAFLD therapy.

Ginsenoside Rg1 protects against H2O2­induced neuronal damage due to inhibition of the NLRP1 inflammasome signalling pathway in hippocampal neurons in vitro.

Oxidative stress and neuroinflammation are important in the pathogenesis of ageing and age­related neurodegenerative diseases, including Alzheimer's disease. NADPH oxidase 2 (NOX2) is a major source of reactive oxygen species (ROS) in the brain. The nucleotide­binding oligomerisation domain (NOD)­like receptor protein 1 (NLRP1) inflammasome is responsible for the formation of pro­inflammatory molecules in neurons. Whether the NOX2­NLRP1 inflammasome signalling pathway is involved in neuronal ageing and age­related damage remains to be elucidated. Ginsenoside Rg1 (Rg1) is a steroidal saponin found in ginseng. In the present study, the primary hippocampal neurons were treated with H2O2 (200 µM) and Rg1 (1, 5 and 10 µM) for 24 h to investigate the protective effects and mechanisms of Rg1 on H2O2­induced hippocampal neuron damage, which mimics age­related damage. The results showed that H2O2 treatment significantly increased ROS production and upregulated the expression of NOX2 and the NLRP1 inflammasome, and led to neuronal senescence and damage to hippocampal neurons. Rg1 decreased ROS production, reducing the expression of NOX2 and the NLRP1 inflammasome in H2O2­treated hippocampal neurons. Furthermore, Rg1 and tempol treatment significantly decreased neuronal apoptosis and the expression of ß­galactosidase, and alleviated the neuronal senescence and damage induced by H2O2. The present study indicates that Rg1 may reduce NOX2­mediated ROS generation, inhibit NLRP1 inflammasome activation, and inhibit neuronal senescence and damage.

Sinomenine exerts anticonvulsant profile and neuroprotective activity in pentylenetetrazole kindled rats: involvement of inhibition of NLRP1 inflammasome.

BACKGROUND: Epilepsy is a common neurological disorder and is not well controlled by available antiepileptic drugs (AEDs). Inflammation is considered to be a critical factor in the pathophysiology of epilepsy. Sinomenine (SN), a bioactive alkaloid with anti-inflammatory effect, exerts neuroprotective activity in many nervous system diseases. However, little is known about the effect of SN on epilepsy. METHODS: The chronic epilepsy model was established by pentylenetetrazole (PTZ) kindling. Morris water maze (MWM) was used to test spatial learning and memory ability. H.E. staining and Hoechst 33258 staining were used to evaluate hippocampal neuronal damage. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: SN (20, 40, and 80 mg/kg) dose-dependently disrupts the kindling acquisition process, which decreases the seizure scores and the incidence of fully kindling. SN also increases the latency of seizure and decreases the duration of seizure in fully kindled rats. In addition, different doses of SN block the hippocampal neuronal damage and minimize the impairment of spatial learning and memory in PTZ kindled rats. Finally, PTZ kindling increases the expression of NLRP1 inflammasome complexes and the levels of inflammatory cytokines IL-1ß, IL-18, IL-6, and TNF-α, which are all attenuated by SN in a dose- dependent manner. CONCLUSIONS: SN exerts anticonvulsant and neuroprotective activity in PTZ kindling model of epilepsy. Disrupting the kindling acquisition, which inhibits NLRP1 inflammasome-mediated inflammatory process, might be involved in its effects.

Acid-sensing ion channels contribute to the effect of extracellular acidosis on proliferation and migration of A549 cells.

Acid-sensing ion channels, a proton-gated cation channel, can be activated by low extracellular pH and involved in pathogenesis of some tumors such as glioma and breast cancer. However, the role of acid-sensing ion channels in the growth of lung cancer cell is unclear. In this study, we investigated the expression of acid-sensing ion channels in human lung cancer cell line A549 and their possible role in proliferation and migration of A549 cells. The results show that acid-sensing ion channel 1, acid-sensing ion channel 2, and acid-sensing ion channel 3 are expressed in A549 cells at the messenger RNA and protein levels, and acid-sensing ion channel-like currents were elicited by extracellular acid stimuli. Moreover, we found that acidic extracellular medium or overexpressing acid-sensing ion channel 1a promotes proliferation and migration of A549 cells. In addition psalmotoxin 1, a specific acid-sensing ion channel 1a inhibitor, or acid-sensing ion channel 1a knockdown can abolish the effect of acid stimuli on A549 cells. In addition, acid-sensing ion channels mediate increase of [Ca2+]i induced by low extracellular pH in A549 cells. All these results indicate that acid-sensing ion channel-calcium signal mediate lung cancer cell proliferation and migration induced by extracellular acidosis, and acid-sensing ion channels may serve as a prognostic marker and a therapeutic target for lung cancer.

Schistosoma japonicum: Tsunagi/Y14 protein plays a critical role in the development of the reproductive organs and eggs.

Tsunagi/Y14 is an evolutionarily conserved RNA-binding protein that is required for the maintenance of oogenesis and the masculinization of the germ-line in many animal models. We speculated that Tsunagi/Y14 might also regulate reproductive organ development in Schistosoma japonicum (S. japonicum, Sj). Sj Tsunagi/Y14 and control double-stranded RNAs were introduced into schistosomula by electroporation respectively. These transfected schistosomula were cultured in vitro for 1, 3 or 5 days. The mRNA and protein levels of the target gene in the cultured schistosomula were significantly suppressed compared with those of the control group. Furthermore, BALB/c mice were infected with the transfected schistosomula for 6 weeks and were sacrificed to harvest the adult worms. We found that the silencing of Sj Tsunagi/Y14 led to defects in reproductive organs development in both male and female worms. Moreover, it also affected the size, quantity and activity of the eggs in the mice liver. Our findings indicated that Tsunagi/Y14 plays a critical role in the development of reproductive organs and eggs in S. japonicum.

[Comparison of collagen fiber staining between Van-Gieson staining and Masson trichrome staining of hepatic specimens in mice with Schistosoma japonicum infection].

OBJECTIVE: To compare the effects of collagen fiber staining between Van-Gieson staining and Masson trichrome staining of hepatic specimens in mice with Schistosoma japonicum infection. METHODS: A model of hepatic granuloma and fibrosis was established by infecting mice with S. japonicum cercariae, then the hepatic specimens were taken and Van-Gieson staining and Masson trichrome staining were performed. Eventually, the area of granuloma and fibrosis were measured by imaging analysis software. RESULTS: When the time of staining was 3-7 min, there was no significant difference of the fibrosis areas between the two methods (P > 0.05); when the time of staining was more than 10 min, the staining area showed by Masson's staining was significantly larger than that showed by Van-Gieson staining, and the difference was statistically significant (P < 0.05). CONCLUSION: The operation procedures of Van-Gieson staining are simpler and easier to master than those of Masson trichrome staining, therefore Van-Gieson staining is a better method to display collagen.

Protective effects of astragalosides on dexamethasone and Aß25-35 induced learning and memory impairments due to decrease amyloid precursor protein expression in 12-month male rats.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder of the elderly characterized by learning and memory impairment. Stress level glucocorticoids (GCs) and ß-amyloid (Aß) peptide deposition are found to be correlated with dementia progression in patients with AD. The astragalosides (AST) was extracted from traditional Chinese herb Astragalus membranaceous. In this study, 12 months male rats were treated with Aß(25-35) (10 µg/rat, hippocampal CA1 injection) and dexamethasone (DEX, 1.5mg/kg, ig) and AST (8, 16 and 32 mg/kg, ig) or ginsenoside Rg1 (Rg1, 5 mg/kg, ig) for 14 days. We investigated the protective effect of AST against DEX+Aß(25-35) injury in rats and its mechanisms of action. Our results indicate that DEX+Aß(25-35) can induce learning and memory impairments and increase APP and Aß(1-40) expression. AST (16, 32 mg/kg) or Rg1 (5mg/kg) treatment significantly improve learning and memory, down-regulate the mRNA levels of APP and ß-secretase, decrease expression of APP and Aß(1-40) in hippocampus. The results indicated that DEX might increase hippocampal vulnerability to Aß(25-35) and highlight the potential neuronal protection of AST.

Worm morphology of Schistosoma japonicum using confocal laser scanning microscopy.

Male and female Schistosoma japonicum worms have dissimilar appearances in their final host. In this study, a morphometric and morphological assessment of whole worms derived from unisexual and mixed infections in mice was conducted using confocal laser scanning microscopy. Worms from mixed infections showed significant morphological changes between 15 and 25 days post-infection (PI). On the fifteenth day PI, 33% of males had formed the conspicuous gynecophoric canal, but only 8% of them had testicular lobes containing a few germinative cells; 13% of females had incipient ovaries with a few immature ovarian cells inside. On the twentieth day PI, the testicular lobes contained more germinative cells in all male worms, while female worms presented vitelline glands. On the twenty-fifth day PI, more germinative cells were observed in the male testicular lobes, and differentiated cells were present in the female ovaries. All worms had fully developed reproductive organs from 30 days PI onwards. Morphometric analysis showed significant differences between mixed and unisexual infections at 35 days PI. Ovaries of worms from unisexual infections contained cells in one stage of maturation and vitelline glands had undifferentiated cells. Our study of S. japonicum provides a detailed comparison of different morphological traits from worms of mixed and unisexual infections throughout development.

Dexamethasone and Aß25-35 accelerate learning and memory impairments due to elevate amyloid precursor protein expression and neuronal apoptosis in 12-month male rats.

Alzheimer's disease (AD) is an irreversible, progressive brain disorder of the elderly characterized by learning and memory impairment. Stress level glucocorticoids (GCs) and ß-amyloid (Aß) peptides deposition are found to be correlated with dementia progression in patients with AD. However, little is known about the simultaneous effects of glucocorticoids and Aß on learning and memory impairment and its mechanism. In this study, 12-month-old male rats were chronically treated with Aß(25-35) (10 µg/rat, hippocampal CA1 injection) and dexamethasone (DEX, 1.5mg/kg) for 14 days to investigate the effects of DEX and Aß(25-35) treatment on learning and memory impairments, pathological changes, neuronal ultrastructure, amyloid precursor protein (APP) processing and neuronal cell apoptosis. Our results showed that DEX or Aß(25-35) treatment alone for 14 days had caused slight damage on learning and memory impairments and hippocampal neurons, but damages were significantly increased with DEX+Aß(25-35) treatment. And the mRNA levels of the APP, ß-secretase and caspase 3 were significantly increased after DEX+Aß(25-35) treatment. The immunohistochemistry demonstrated that APP, Aß(1-40), caspase 3 and cytochrome c in hippocampus CA1 were significantly increased. Furthermore, Hoechst 33258 staining and Aß(1-40) ELISA results showed that DEX+Aß(25-35) treatment induced hippocampus CA1 neuron apoptosis and increased the level of Aß(1-40). The results suggest that the simultaneous effects of GCs and Aß may have important roles in the etiopathogenesis of AD, and demonstrate that stressful life events and GC therapy may increase the toxicity of Aß and have cumulative impacts on the course of AD development and progression.

[Preliminary function study on Mago nashi gene of Schistosoma japonicum].

OBJECTIVE: To study the function of Mago nashi gene in reproductive system of Schistosoma japonicum. METHODS: dsRNA products of SjMago nashi gene and control gene (lacZ) were generated by in vitro transcription. SjMago nashi dsRNA and control (lacZ) dsRNA were electroporated into mechanically transformed schistosomula. Aliquots of parasites (1000) were harvested at day 1, 3, and 5 after electroporation, respectively. Total RNA and proteins were isolated simultaneously using TRIzol reagent. Levels of SjMago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis, respectively. About 1000 dsRNA-electroporated schistosomula were injected into each BALB/c female mouse. Six weeks later the worms were collected, fixed, stained, clarified, dehydrated and mounted. The male and female reproductive organs were observed and measured under the confocal laser scanning microscope. RESULTS: At day 1, 3 and 5 post-electroporation, 22%, 69%, and 80% reduction in Mago nashi mRNA levels were detected respectively in SjMago nashi dsRNA-electroporated schistosomula (experiment group) compared to parasites treated with control dsRNA (control group); and schistosomula of experiment group exhibited 12%, 39%, and 56% decreased in Mago nashi protein expression levels in comparison to the control group, respectively. In experiment group there were many spermatozoa in testicular lobes and no changes were observed in ovary and vitelline gland. Compared to control group, adult worms in experiment group were smaller in the body width, the width and length of testicles and ovaries (P < 0.05). CONCLUSION: Mago nashi dsRNA can specifically inhibit the expression of target gene and protein. SjMago nashi gene is a reproduction-related gene.

Expression of cell proliferation and apoptosis biomarkers in pterygia and normal conjunctiva.

PURPOSE: To analyze the expression of apoptosis and cell proliferation molecules in pterygium tissues of Chinese patients. METHODS: Thirty-three pterygia were surgically removed using the bare sclera procedure, and 23 normal bulbar conjunctivas were also obtained. Formalin-fixed, paraffin-wax-embedded tissues were analyzed by immunohistochemistry with anti- proliferating cell nuclear antigen (PCNA), K(i)-67 (a proliferating cell marker), mutant p53 (mP53), Bcl-2 associated X-protein (BAX), B-cell lymphoma gene 2 (Bcl-2), and caspase-3 antibodies. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) analysis was used to analyze the apoptotic cells. RESULTS: Our study revealed that the positive rate of PCNA and K(i)-67 significantly increased in the pterygium samples compared to the normal conjunctiva samples. In the molecules involved in apoptosis, the results showed that the positive rate of Bcl-2 and mP53 significantly increased in the pterygium samples. However, no difference was found between the pterygium and normal conjunctiva samples in the expression of Bax and caspase-3. Through TUNEL analysis, apoptotic cells were seen in the entire width of the epithelial layer in normal conjunctivas but were found mainly confined to the outer layer of the epithelial cells in pterygia. CONCLUSIONS: The finding of high levels of cellular proliferation and low levels of cellular apoptosis in pterygia confirmed that both cell apoptosis and proliferation are known to play an important role in human pterygium pathogenesis.

[Morphological observation on the reproductive system of adult Schistosoma japonicum under confocal laser scanning microscopy].

BALB/c mice were infected with Schistosoma japonicum cercariae (40+/-2 per mouse) through abdominal skin. Mice were sacrificed after 35 days to acquire the adult worms which were then fixed, stained, clarified, dehydrated and mounted. The specimens were observed under the confocal laser scanning microscope. The overall morphology of the adult worms was displayed distinctly, especially the testicular lobes, seminal vesicle and genital pore of the male, reproductive system, and the ovary, vitelline glands, oviduct, vitelline duct, seminal receptacle, ootype, mehlis gland, uterus, genital pore and eggs of female reproductive system. The confocal laser scanning microscopy is an alternative method to research organs, tissues and cell structure of schistosome worm.