Hypoglycemic mechanism of Tegillarca granosa polysaccharides on type 2 diabetic mice by altering gut microbiota and regulating the PI3K-akt signaling pathwaye

Type 2 diabetes mellitus(T2DM)is a complex metabolic disease threatening human health.We investigated the effects of Tegillarca granosa polysaccharide(TGP)and determined its potential mechanisms in a mouse model of T2DM established through a high-fat diet and streptozotocin.TGP(5.1×10^(3) Da)was composed of mannose,glucosamine,rhamnose,glucuronic acid,galactosamine,glucose,galactose,xylose,and fucose.It could significantly alleviate weight loss,reduce fasting blood glucose levels,reverse dyslipidemia,reduce liver damage from oxidative stress,and improve insulin sensitivity.RT-PCR and Western blotting indicated that TGP could activate the phosphatidylinositol-3-kinase/protein kinase B signaling pathway to regulate disorders in glucolipid metabolism and improve insulin resistance.TGP increased the abundance of Allobaculum,Akkermansia,and Bifidobacterium,restored the microbiota abundance in the intestinal tracts of mice with T2DM,and promoted short-chain fatty acid production.This study provides new insights into the antidiabetic effects of TGP and highlights its potential as a natural hypoglycemic nutraceutical.

“Baihui”(DU20)-penetrating “Qubin”(GB7) acupuncture on blood–brain barrier integrity in rat intracerebral hemorrhage models via the RhoA/ROCK Ⅱ/MLC 2 signaling pathway

Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.

Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Naringin ameliorates H_(2)O_(2)-induced oxidative damage in cells and prolongs the lifespan of female Drosophila melanogaster via the insulin signaling pathway

Naringin exists in a wide range of Chinese herbal medicine and has proven to possess several pharmacological properties.In this study,PC12,HepG2 cells,and female Drosophila melanogaster were used to investigate the antioxidative and anti-aging effects of naringin and explore the underlying mechanisms.The results showed that naringin inhibited H_(2)O_(2)-induced decline in cell viability and decreased,the content of reactive oxygen species in cells.Meanwhile,naringin prolonged the lifespan of flies,enhanced the abilities of climbing and the resistance to stress,improved the activities of antioxidant enzymes,and decreased malondialdehyde content.Naringin also improved intestinal barrier dysfunction and reduced abnormal proliferation of intestinal stem cells.Moreover,naringin down-regulated the mRNA expressions of inr,chico,pi 3k,and akt-1,and up-regulated the mRNA expressions of dilp2,dilp3,dilp5,and foxo,thereby activating autophagy-related genes and increasing the number of lysosomes.Furthermore,the mutant stocks assays and computer molecular simulation results further indicated that naringin delayed aging by inhibiting the insulin signaling(IIS)pathway and activating the autophagy pathway,which was consistent with the result of network pharmacological predictions.

X-Paste improves wound healing in diabetes via NF-E2-related factor/HO-1 signaling pathway

BACKGROUND Diabetic foot ulcers(DFU),as severe complications of diabetes mellitus(DM),significantly compromise patient health and carry risks of amputation and mortality.AIM To offer new insights into the occurrence and development of DFU,focusing on the therapeutic mechanisms of X-Paste(XP)of wound healing in diabetic mice.METHODS Employing traditional Chinese medicine ointment preparation methods,XP combines various medicinal ingredients.High-performance liquid chromatography(HPLC)identified XP’s main components.Using streptozotocin(STZ)-induced diabetic,we aimed to investigate whether XP participated in the process of diabetic wound healing.RNA-sequencing analyzed gene expression differences between XP-treated and control groups.Molecular docking clarified XP’s treatment mechanisms for diabetic wound healing.Human umbilical vein endothelial cells(HUVECs)were used to investigate the effects of Andrographolide(Andro)on cell viability,reactive oxygen species generation,apoptosis,proliferation,and metastasis in vitro following exposure to high glucose(HG),while NF-E2-related factor-2(Nrf2)knockdown elucidated Andro’s molecular mechanisms.RESULTS XP notably enhanced wound healing in mice,expediting the healing process.RNA-sequencing revealed Nrf2 upregulation in DM tissues following XP treatment.HPLC identified 21 primary XP components,with Andro exhibiting strong Nrf2 binding.Andro mitigated HG-induced HUVECs proliferation,metastasis,angiogenic injury,and inflammation inhibition.Andro alleviates HG-induced HUVECs damage through Nrf2/HO-1 pathway activation,with Nrf2 knockdown reducing Andro’s proliferative and endothelial protective effects.CONCLUSION XP significantly promotes wound healing in STZ-induced diabetic models.As XP’s key component,Andro activates the Nrf2/HO-1 signaling pathway,enhancing cell proliferation,tubule formation,and inflammation reduction.

Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

Objective:To investigate the mechanism of JAK2/STAT3/SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods:36 SPF SD rats were randomly divided into 6 groups,with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group,with density wave,frequency 2Hz-50 Hz,intensity 2 mA,muscle tremor as the degree 20 min/time,1 time/day,for 3 consecutive days.Observe the general condition of rats;the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4(IL-4)and interleukin-8(IL-8)were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2,STAT3,SOCS1 protein and mRNA in rat colon tissue.Results:In contrast to the normal group,the overall condition of the model group was worse,the colonic mucosa was severely damaged,even necrotic,and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced,and the content of IL-8 was obviously go up(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously go up,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced(P<0.01).In contrast to the model group,the general condition of rats in each acupoint group was significantly improved,the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated,the content of IL-4 in serum was obviously go up,and the content of IL-8 was significantly decreased(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously reduced,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up(P<0.05,P<0.01).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced,the content of serum IL-4 was significantly increased,and the content of IL-8 was significantly decreased(P<0.05,P<0.01).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated,while the protein content and mRNA expression of SOCS1 were significantly go up(P<0.05,P<0.01).Conclusion:Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli(ST36)is better than that of Tianshu(ST25),Dachangshu(BL25)and Shangjuxu(ST37).The mechanism may be related to the regulation of JAK2/STAT3/SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

Oleanolic acid inhibits colon cancer cell stemness and reverses chemoresistance by suppressing JAK2/STAT3 signaling pathway

Background:Oleanolic acid(OA),a pentacyclic triterpenoid exhibiting specific anti-cancer properties and highly effective antioxidant activity,was isolated from traditional Chinese medicinal herbs.Conversely,the OA that impacts colon cancer(CC)cells and its underlying mechanisms remain poorly understood.Methods:The cytotoxic effect of OA alone or OA-5-Fluorouracil(5-FU)combination on normal and CC cells was analyzed by methyl thiazolyl diphenyl-tetrazolium bromide(MTT).Then,the impact of OA on CC cell lines(LoVo and HT-29)proliferation and stemness were measured using colon formation and tumorsphere formation assays.Octamer-binding transcription factor 4(Oct4),Prominin-1(CD133),Nanog,and transcription factor SOX-2(SOX2)are cell stemness-related indicators whose expression was assessed usingfluorescence qPCR assay,Western blotting,and immunohistochemistry.The effect of OA on the proliferative potency of CC cells was evaluated using an in vivo model.Results:The stem-like characteristics and clone production of colon cancer cells were markedly reduced by OA alone or in combination with OA-5-FU.Moreover,OA increases the susceptibility of CC cells to 5-FU by blocking the cell stemness-related markers(CD133,Nanog,SOX2,and Oct4)expression levels both in vitro and in vivo,as well as by inactivating the activator of transcription 3(STAT3 signaling)and Janus kinase 2/signal transducer(JAK2).Conclusion:Thesefindings imply that oleanolic acid,both in vitro and in vivo,suppresses the JAK2/STAT3 pathway,which in turn reverses chemoresistance and decreases colon cancer cell stemness.Therefore,by reducing the recommended amount of 5-FU,this strategy may improve chemotherapeutic effectiveness and minimize undesired side effects.

Comparative study of anti-inflammatory effects of different processed products through the COX-2/PGE2 signaling pathway: based on network pharmacology and molecular docking

Background:Radix Aconiti Lateralis Preparata(Fu-zi)is a traditional Chinese medicinal herb,which has been widely used in the clinic and has potent anti-inflammatory activities.we aimed to explore the mechanisms of extract containing alkaloids from different Fu-zi Processed Products(FPP)in treating inflammation,especially rheumatoid arthritis(RA).Methods:Firstly,using network pharmacology technology,the ingredients,and targets of Fu-zi were obtained by searching and screening,the targets involving RA were acquired,the intersection targets were constructed a"component-target-pathway"network.A comprehensive investigation was conducted on the anti-rheumatoid arthritis mechanisms of 5 FPPs in lipopolysaccharide(LPS)induced RAW264.7 cells,which serve as a model for RA.The production of NO and inflammatory cytokines were measured by ELISA kit.Quantitative Real-time PCR(qRT-PCR)was utilized to measure the mRNA levels.COX-2/PGE2 signaling pathway-associated proteins were determined by western blot.Results:According to a network pharmacological study,16 chemical components and 43 common targets were found in Fu-zi and 6 key targets including PTGS2 were closely related to the mechanism of Fu-zi in treating RA.The in vitro study revealed that the levels of NO,TNF-α,and IL-1βwere substantially decreased by the 5 FPPs.The 5 FPPs significantly suppressed the expression of proteins COX-2,iNOS,and NF-κB,with particularly notable effects observed for PFZ and XFZ.Conclusion:Altogether,these results demonstrated that the 5 PPS containing alkaloids have a good anti-RA-related inflammatory effect,and the mechanism may be related to COX-2/PGE2 signaling pathway,particularly,Fu-zi prepared utilizing a traditional Chinese technique.

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway

The current study aimed to assess the effect of timosaponin AⅢ(T-AⅢ)on drug-metabolizing enzymes during anticancer therapy.The in vivo experiments were conducted on nude and ICR mice.Following a 24-day administration of T-AⅢ,the nude mice exhibited an induction of CYP2B10,MDR1,and CYP3A11 expression in the liver tissues.In the ICR mice,the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration.The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6,MDR1,and CYP3A4,along with constitutive androstane receptor(CAR)activation.Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression.Furthermore,other CAR target genes also showed a significant increase in the expression.The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice.Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation,with this effect being partially reversed by the ERK activator t-BHQ.Inhibition of the ERK1/2 signaling pathway was also observed in vivo.Additionally,T-AⅢ inhibited the phosphorylation of EGFR at Tyr1173 and Tyr845,and suppressed EGF-induced phosphorylation of EGFR,ERK,and CAR.In the nude mice,T-AⅢ also inhibited EGFR phosphorylation.These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

Reduction of the oxidative damage to H_(2)O_(2)-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside

Hyperoside and quercetin are similar in molecular structures.In this study,the antioxidant regulatory targets of hyperoside and quercetin are mainly in the nuclear factor(erythroid-2-derived)-related factor 2(Nrf2)pathway predicted by network pharmacology.And the antioxidant effect and mechanism of hyperoside and quercetin were measured and compared in H_(2)O_(2)-induced Hep G2 cells and Caenorhabditis elegans.The findings indicated that quercetin was more effective than hyperoside in reducing oxidative damage,which was proved by improved cell viability,decreased reactive oxygen species(ROS)production,decreased cellular apoptosis,and alleviated mitochondrial damage.In addition,quercetin was more efficient than hyperoside in enhancing the expression of Nrf2-associated m RNAs,increasing the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT),and reducing the cellular malondialdehyde(MDA)content.Quercetin was superior to hyperoside in prolonging the lifespan of worms,decreasing the accumulation of lipofuscin,inhibiting ROS production,and increasing the proportion of skn-1 in the nucleus.With the Nrf2 inhibitor ML385,we verified that quercetin and hyperoside primarily protected the cells against oxidative damage via the Nrf2 signalling pathway.Furthermore,molecular docking and dynamics simulations demonstrated that the quercetin-Kelch-like ECH-associated protein 1(Keap1)complex was more stable than the hyperoside-Keap1 complex.The stable structure of the complex might hinder the binding of Nrf2 and Keap1 to release Nrf2 and facilitate its entry into the nucleus to play an antioxidant role.Overall,quercetin had a better antioxidant than hyperoside.

CK2αcauses stemness and chemotherapy resistance in liver cancer through the Hedgehog signaling pathway

Background:Liver cancer is one of the major causes of cancer-related deaths globally.Cancer cell stem-ness and chemotherapy resistance contribute to the high mortality.Although evidence indicates that the alpha subunit of protein kinase 2(CK2α)is involved in several human cancers,its function in liver cancer remains unknown.In the present study,we aimed to elucidate the role of CK2αin liver cancer.Methods:We examined the role of CK2αregulation in stemness and chemotherapy resistance capacity of liver cancer cells.MTT assays,tumor sphere formation assays,RT-PCR,flow cytometry,Western blotting assay,clonogenicity assay,matrigel invasion assay and bioinformatics were conducted in this study.Results:CK2αexpression in the liver cancer tissues was notably upregulated compared with that in the corresponding non-tumorous tissues.The overexpression of CK2αpromoted tumor sphere formation,increased the percentage of CD133(+)and side population cells,caused the resistance of liver cancer cells to 5-FU treatment,increased the expression levels of NANOG,OCT4,SOX2,Gli1 and Ptch1,and enhanced the ability of CD133(+)cell clone formation and invasion.Consistently,the downregulation of CK2αhad the opposite effects.CK2αsilencing inhibited the Hedgehog pathway by reducing the expression of Gli1 and Ptch1.Mechanistically,CK2αregulation on liver cancer cell stemness and chemotherapy resistance was found to be involved in the Hedgehog signaling pathway.Conclusions:Our study may bring some new insights into the occurrence of liver cancer.Furthermore,these findings suggest that targeting CK2αmay be a novel therapeutic strategy for patients with liver cancer.

Adenosine 2A receptor contributes to the facilitation of postinfectious irritable bowel syndrome by γδ T cells via the PKA/CREB/NF-κB signaling pathway

BACKGROUND Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome(PI-IBS).γδT cells play a crucial role in innate and adaptive immunity.Adenosine receptors expressed on the surface ofγδT cells participate in intestinal inflammation and immunity regulation.AIM To investigate the role ofγδT cell regulated by adenosine 2A receptor(A2AR)in PI-IBS.METHODS The PI-IBS mouse model has been established with Trichinella spiralis(T.spiralis)infection.The intestinal A2AR and A2AR inγδT cells were detected by immunohistochemistry,and the inflammatory cytokines were measured by western blot.The role of A2AR on the isolatedγδT cells,including proliferation,apoptosis,and cytokine production,were evaluated in vitro.Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction(RT-PCR).The animals were administered with A2AR agonist,or A2AR antagonist.Besides,γδT cells were also injected back into the animals,and the parameters described above were examined,as well as the clinical features.Furthermore,the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR.RESULTS PI-IBS mice exhibited elevated ATP content and A2AR expression(P<0.05),and suppression of A2AR enhanced PI-IBS clinical characteristics,indicated by the abdominal withdrawal reflex and colon transportation test.PI-IBS was associated with an increase in intestinal T cells,and cytokine levels of interleukin-1(IL-1),IL-6,IL-17A,and interferon-α(IFN-α).Also,γδT cells expressed A2AR in vitro and generated IL-1,IL-6,IL-17A,and IFN-α,which can be controlled by A2AR agonist and antagonist.Mechanistic studies demonstrated that the A2AR antagonist improved the function ofγδT cells through the PKA/CREB/NF-κB signaling pathway.CONCLUSION Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function ofγδT cells via the PKA/CREB/NF-κB signaling pathway.

Effects of plumbagin on migration and invasion of human hepatoma cell line via JAK2/STAT3 signaling pathway

Objective:To study the effect of plumbagin(PL)on the migration and invasion of human hepatocellular carcinoma(HCC)cells and its possible mechanism.Methods:The cell counting kit(CCK-8)was used to detect the effects of different concentrations of plumbagin on the proliferation of human hepatocellular carcinoma Huh-7 and LM3 cells.The effect of plumbagin on the migration ability of Huh-7 and LM3 cells was detected by scratch test and Transwell migration test,and the effect of on the invasion ability of Huh-7 and LM3 cells was detected by Transwell invasion test.Western Blot was used to detect the expression of E-cadherin,N-cadherin,matrix metalloproteinase-2 and related proteins in JAK2/STAT3 signaling pathway in Huh-7 and LM3 cells.Results:Plumbagin could inhibit the proliferation of Huh-7 and LM3 cells in a time-and concentration-dependent manner.Plumbagin inhibited the migration and invasion of Huh-7 and LM3 cells in a concentration dependent manner,and it can down-regulate the expression of N-cadherin and MMP-2 protein,up-regulate the expression of E-cadherin protein,and inhibit the activation of JAK2/STAT3 signaling pathway.Conclusion:Plumbagin can inhibit the migration and invasion of human hepatocellular carcinoma Huh-7 and LM3 cells,and the molecular mechanism of this process may be related to the inhibition of JAK2/STAT3 signaling pathway activation.

Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

Anti-diabetic potential of apigenin,luteolin,and baicalein via partially activating PI3K/Akt/GLUT-4 signaling pathways in insulin-resistant HepG2 cells

Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant(IR)HepG2 cells.All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4(GLUT4)and phosphor-glycogen synthase kinase(GSK-3β).These fl avonoids signifi cantly inhibited the production of reactive oxygen species(ROS)and advanced glycation end-products(AGEs),which were closely related to the suppression of the phosphorylation form of NF-κB and P65.The expression levels of insulin receptor substrate-1(IRS-1),insulin receptor substrate-2(IRS-2)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway in IR-HepG2 cells were all partially activated by the fl avonoids,with variable effects.Furthermore,the intracellular metabolic conditions of the fl avonoids were also evaluated.

High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway

AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous protein(CHOP)-cysteine aspartate specific proteinase(caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells(RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48 h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 k Da glucose-regulated protein 78(GRP78), CHOP, B-cell lymphoma 2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eI F2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs(P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12(P<0.05), and the alterations caused by glucose were in concentration-and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups(P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group(P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits(P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eI F2α in the HCG group(P<0.05), while still did not affect the expression of PERK and eIF2α among groups(P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose(P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOPcaspase-12 signaling pathway and promotes apoptosis of RCECs.

Melatonin improves synapse development by PI3K/Akt signaling in a mouse model of autism spectrum disorder

Autism spectrum disorders are a group of neurodevelopmental disorders involving more than 1100 genes,including Ctnnd2 as a candidate gene.Ctnnd2knockout mice,serving as an animal model of autis m,have been demonstrated to exhibit decreased density of dendritic spines.The role of melatonin,as a neuro hormone capable of effectively alleviating social interaction deficits and regulating the development of dendritic spines,in Ctnnd2 deletion-induced nerve injury remains unclea r.In the present study,we discove red that the deletion of exon 2 of the Ctnnd2 gene was linked to social interaction deficits,spine loss,impaired inhibitory neurons,and suppressed phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt) signal pathway in the prefrontal cortex.Our findings demonstrated that the long-term oral administration of melatonin for 28 days effectively alleviated the aforementioned abnormalities in Ctnnd2 gene-knockout mice.Furthermore,the administration of melatonin in the prefro ntal cortex was found to improve synaptic function and activate the PI3K/Akt signal pathway in this region.The pharmacological blockade of the PI3K/Akt signal pathway with a PI3K/Akt inhibitor,wo rtmannin,and melatonin receptor antagonists,luzindole and 4-phenyl-2-propionamidotetralin,prevented the melatonin-induced enhancement of GABAergic synaptic function.These findings suggest that melatonin treatment can ameliorate GABAe rgic synaptic function by activating the PI3K/Akt signal pathway,which may contribute to the improvement of dendritic spine abnormalities in autism spectrum disorders.

Abnormally activated wingless/integrated signaling modulates tumor-associated macrophage polarization and potentially promotes hepatocarcinoma cell growth

In this article,we comment on the article by Huang et al.The urgent development of new therapeutic strategies targeting macrophage polarization is critical in the fight against liver cancer.Tumor-associated macrophages(TAMs),primarily of the M2 subtype,are instrumental in cellular communication within the tumor microenvironment and are influenced by various signaling pathways,including the wingless/integrated(Wnt)pathway.Activation of the Wnt signaling pathway is pivotal in promoting M2 TAMs polarization,which in turn can exacerbate hepatocarcinoma cell proliferation and migration.This manuscript emphasizes the burgeoning significance of the Wnt signaling pathway and M2 TAMs polarization in the pathogenesis and progression of liver cancer,highlighting the potential therapeutic benefits of inhibiting the Wnt pathway.Lastly,we point out areas in Huang et al’s study that require further research,providing guidance and new directions for similar studies.

β-arrestin 2 attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation in mice

AIM To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide(LPS)-induced liver injury in vivo and in vitro.METHODS Male β-arrestin 2^(+/+) and β-arrestin 2^(-/-)C57 BL/6 J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.RESULTS Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of proinflammatory cytokines, including interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines(including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 si RNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phosphoIκBα and phosho-p65, were upregulated.CONCLUSION β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation.