Antifertility activity of Oroxylum indicum (L.) Kurz:In vitro and in vivo study on human sperm and male wistar rats

Oroxylum indicum (L.) Kurz, commonly called as Broken bones tree or Indian trumpet flower, belonging to Fam. Bignoniaceae, is traditionally used as a contraceptive by ethnic people of Tripura, North-East India. Here, we investigated the scientific basis for use of O. indicum as male antifertility agent by folklore healers. In vitro spermicidal activity of aqueous (AEOI) and methanolic (MEOI) extracts of O. indicum stem bark were studied on human sperm. The in vivo activity was experimented on male albino rats. The treated animals were allowed to mate and the pups delivered by female rat partners were counted. Phytochemical estimation of test samples was done using HPLC. The AEOI and MEOI treatments significantly decreased human sperm motility and viability. Test extracts have increased the hypo-osmotic swelling of sperm. Both the extracts were significantly declined the weight of reproductive organ. The MEOI treated rats have shown significant decrease in sperm motility and sperm counts. AEOI and MEOI treatment significantly reduced level of testosterone, but sharply raised dihydrotestosterone and prostaglandin in rats. Results testified the traditional claim for use of O. indicum as a male contraceptive agent, where MEOI have shown reversible action on male reproductive system leading to contraception without harming the libido.

Optimization of preparation technology and in vitro release of baicalin-glycyrrhizic acid solid nanocrystals / 中国药房

OBJECTIVE To optimize the preparation technology of baicalin （BCN）-glycyrrhizic acid （GA） solid nanocrystals （BCN-GA-SN）， to characterize them and investigate their in vitro release characteristics. METHODS According to the compatibility ratio of classic couplet medicinals “Scutellaria baicalensis-Glycyrrhiza uralensis”， the compatibility ratio of BCN and GA was determined as 6∶1 （m/m）； BCN-GA nanosuspension was prepared by precipitation method combined with high-pressure homogenization method. The preparation technology of BCN-GA nanosuspension was optimized by using mean particle size and polydispersity index （PDI） as indexes and with types and dosage of stabilizers， stirring speed and time， high-pressure homogenization pressure and frequency as factors. The freeze-dried consolidation process of BCN-GA nanosuspension was optimized to prepare BCN-GA-SN using average particle size， PDI and redispersibility index （RDI） as indicators， with the type and dosage of freeze-dried protective agents as factors； then， the physicochemical properties and in vitro release of BCN-GA-SN were investigated. RESULTS The optimal preparation technology of BCN-GA-SN was as follows： BCN-GA nanosuspension was prepared by using 15% sodium dodecyl sulfate as a stabilizer， stirring at 1 000 r/min for 15 minutes， and homogenizing at 100 MPa for 20 times； then， BCN-GA nanosuspension was freeze-dried and solidified with 5% mannitol （corresponding to the dosage of BCN）. The average particle size of prepared BCN-GA-SN was （442.2±5.7） nm with PDI of 0.225±0.015 and RDI of 1.055± 0.013. The prepared BCN-GA-SN presented as the irregularly spherical shape with more uniform size； the drug-loading amount of BCN in the nanocrystal was （62.5±0.7）%， and that of GA was （9.4±0.2）%； the in vitro release results showed that the cumulative dissolution of BCN-GA-SN was higher than that of the physical mixture of BCN and GA. CONCLUSIONS BCN-GA-SN is prepared successfully in this study with uniform particle size and even distribution， which can effectively improve the dissolution of BCN.

Baicalin Antagonizes Prostate Cancer Stemness via Inhibiting Notch1/NF-κB Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanisms underlying the effect of baicalin on prostate cancer (PCa) progression both in vivo and in vitro.@*METHODS@#The in situ PCa stem cells (PCSCs)-injected xenograft tumor models were established in BALB/c nude mice. Tumor volume and weight were respectively checked after baicalin (100 mg/kg) treatment. Hematoxylin-eosin (HE) staining was used to observe the growth arrest and cell necrosis. mRNA expression levels of acetaldehyde dehydrogenase 1 (ALDH1), CD44, CD133 and Notch1 were determined by reverse transcription-polymerase chain reaction. Protein expression levels of ALDH1, CD44, CD133, Notch1, nuclear factor κB (NF-κB) P65 and NF-κB p-P65 were detected by Western blot. Expression and subcellular location of ALDH1, CD44, CD133, Notch1 and NF-κB p65 were detected by immunofluorescence analysis. In vitro, cell cycle distribution and cell apoptosis of PC3 PCSCs was assessed by flow cytometry after baicalin (125 µmol/L) treatment. The migration and invasion abilities of PCSCs were assessed using Transwell assays. Transmission electron microscopy scanning was utilized to observe the structure and autophagosome formation of baicalin-treated PCSCs. In addition, PCSCs were infected with lentiviruses expressing human Notch1.@*RESULTS@#Compared with the control group, the tumor volume and weight were notably reduced in mice treated with 100 mg/kg baicalin (P<0.05 or P<0.01). Histopathological analysis showed that baicalin treatment significantly inhibited cell proliferation and promoted cell apoptosis. Furthermore, baicalin treatment reduced mRNA and protein expression levels of CD44, CD133, ALDH1, and Notch1 as well as the protein expression of NF-κB p-P65 in the xenograft tumor (P<0.01). In vitro, the cell proliferation of PCSCs was significantly attenuated after treatment with 125 µmol/L baicalin for 72 h (P<0.01). The cell migration and invasion rates were decreased following treatment with baicalin for 48 and 72 h (P<0.01). Baicalin notably induced cell apoptosis and seriously damaged the structure of PCSCs. The mRNA and protein expressions of CD133, CD44, ALDH1 and Notch1 in PCSCs were significantly downregulated following baicalin treatment (P<0.01). Importantly, the inhibitory effects of baicalin on PCa progression and stemness were reversed by Notch1 overexpression (P<0.05 or P<0.01).@*CONCLUSION@#Mechanistically, baicalin exhibited a potential therapeutic effect on PCa via inhibiting the Notch1/NF-κB signaling pathway and its mediated cancer stemness.

Baicalin Induces Ferroptosis in Gastric Cancer Cells via p53-mediated SLC7A11 Down-regulation / 中国实验方剂学杂志

ObjectiveTo explore the inhibitory effect of different concentration of baicalin （0， 100， 200， 400 μmol·L-1） on the proliferation of human gastric cancer SGC-7901 cells and the underlying mechanism. MethodSGC-7901 cells were treated with baicalin. Then methyl thiazolyl tetrazolium （MTT） assay was employed to examine the inhibitory effect of baicalin on the cells. At the same time， ferrostatin-1 （Fer-1） was added to observe the viability of cells after baicalin treatment. The expression of ferroptosis-related genes was detected by Real-time polymerase chain reaction （Real-time PCR） and Western blot. The content of malondialdehyde （MDA） and the level of glutathione （GSH） were detected respectively by MTT assay and enzyme-linked immunosorbent assay. The role of tumor protein 53 （p53）/solute carrier family 7 member 11 （SLC7A11） pathway in the regulation of ferroptosis was investigated respectively via overexpression and small interfering RNA （siRNA） methods. ResultCompared with the blank group， baicalin decreased the viability of SGC-7901 （P<0.05， P<0.01） in a dose- and time-dependent manner. The intervention of Fer-1 significantly alleviated the decrease of SGC-7901 cell viability caused by baicalin （P<0.01）. In addition， compared with the baicalin group， Fer-1+baicalin group showed decrease in MDA content and the mRNA and protein levels of prostaglandin-endoperoxide synthase 2 （PTGS2） in the cells （P<0.01）， and increase in GSH activity and mRNA and protein levels of glutathione peroxidase 4 （GPX4） （P<0.01）. The protein level of SLC7A11 in the baicalin group was decreased compared with that in the blank group （P<0.05， P<0.01） in a dose-dependent manner. Compared with the baicalin group， the reactive oxygen species （ROS） level and MDA content in SLC7A11-overexpressing cells were significantly decreased after baicalin treatment （P<0.01）， and the GSH activity was significantly increased （P<0.01）. The fluorescence intensity of p53 in the cells of the baicalin group was increased compared with that of the blank group （P<0.01）. Compared with the baicalin group， the expression level of p53 protein in the cells transfected with p53 siRNA was significantly decreased after baicalin treatment （P<0.01）， and the expression level of SLC7A11 was significantly increased （P<0.01）. ConclusionBaicalin can effectively inhibit the proliferation of SGC-7901 cells by regulating p53/SLC7A11-mediated ferroptosis.

Baicalin inhibits PDK1 to mediate glucose metabolism reprogramming and intervene rheumatoid arthritis synovial inflammation / 药学学报

This study started from the effect of baicalin (BC), the main active component of the labiaceae plant Scutellaria baicalensis, on collagen-induced arthritis (CIA) in rats, to explore the mechanism of glucose metabolism reprogramming in fibroblast like synoviocytes (FLSs), a key effector cell of synovial inflammation in rheumatoid arthritis (RA). First of all, CIA rats and tumor necrosis factor-α (TNF-α)-induced RASFs in vitro and in vivo models were established, the arthritis index (AI) score and histopathological changes of CIA rats after BC administration were observed, and the levels of inflammatory factors in serum and cell supernatant were quantified by ELISA, immunocytochemistry and Western blot were used to detect the expression of G-protein-coupled receptor 81 (GPR81) and pyruvate dehydrogenase kinase 1 (PDK1) proteins. In addition, the kit was used to measure the levels of key products and enzyme activities in glucose metabolism reprogramming. The results showed that BC (50, 100 and 200 mg·kg-1) could alleviate the symptoms of arthritis in CIA rats in a dose-dependent manner, inhibit synovial hyperplasia, alleviate the infiltration of inflammatory cells, down-regulate the levels of pro-inflammatory factors TNF-α and interleukin (IL)-1β, and up-regulate the levels of anti-inflammatory factor IL-10 in CIA rats. At the same time, the secretion levels of lactate, pyruvate, acetyl-CoA, citrate and the activity of lactate dehydrogenase B (LDH-B) were decreased, and the expressions of GRP81 and PDK1 were down-regulated, suggesting that BC mediated the reprogramming process of glucose metabolism. However, when GPR81 inhibitor 3-OBA inhibited lactate uptake, the activity of LDH-B was significantly increased, suggesting that BC inhibited the expression of PDK1, a key enzyme in the reprogramming metabolism from glycolysis to oxidative phosphorylation. All animal experiments in this study were conducted in accordance with the ethical standards of the Laboratory Animal Care Center of Anhui University of Chinese Medicine (approval number: AHUCM-rats-2021049). These studies revealed that baicalin mediated metabolic reprogramming of RASFs from glycolysis to oxidative phosphorylation by inhibiting PDK1 protein expression, and alleviated joint inflammation in CIA rats.

Cellular metabolomic study of baicalin against respiratory syncytial virus / 中国药理学通报

Aim To explore the effect of baicalin on respiratory syncytial virus in vitro and its effect on cell metabolism. Methods The anti-RSV effect of baicalin in vitro was verified by antiviral cell experiment, and the cellular metabolic mechanism of baicalin against RSV was explored by cell metabolomics. Results Baicalin had an inhibitory effect on all stages of RSV infection, and the condition of CPE was significantly improved, which may mainly play a role in the adsorption and proliferation of RSV. A total of 19 differential metabolites were screened by cell metabolomics, which were mainly glycerol phospholipids, nucleosides and fatty acids. Seven metabolic pathways were obtained by enrichment analysis, which were mainly related to glycerol-phospholipid metabolism, fatty acid metabolism (arachidonic acid metabolism, α-linolenic acid metabolism, linoleic acid metabolism), amino acid metabolism and purine metabolism. Conclusions Baicalin has significant inhibitory effect on the adsorption and proliferation of RSV, which may be related to fatty acid metabolism, glycerol phospholipid metabolism, amino acid and purine metabolism.

Baicalin inhibits streptozotocin-induced neuroinflammation in Alzheimer' s disease rat model by TLR4/MyD88/NF-κB pathway / 中国药理学通报

Aim To investigate the effects of baicalin on the inflammatory response and Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD 88)/nuclear factor kappa B (N F-K B) signaling pathway in Alzheimer' s disease (AD) rat model induced by lateral ventricular injection of streptozotocin (STZ). Methods The AD animal model was constructed by lateral ventricular injection of STZ in SD rats, and divided into sham operation group, model group, low-dose (60 mg

Baicalin modulates let-7i-3p/PI3K/Akt/NF-KB signaling axis to reduce NLRP3 inflammasome activation in fibroblast like synoviocytes of rheumatoid arthritis / 中国药理学通报

Aim To study the effect of baicalin on the activation of NLRP3 inflammasomes in human fibroblast like synoviocytes of rheumatoid arthritis ( HFLS-RA) and its mechanism. Methods To confirm that baicalin alleviated the activation of NLRP3 inflammasome in HFLS-RA, immunofluorescence was used to observe the expression of NLRP3 before and after baicalin treatment. Western blot was used to detect the protein expression of p-PI3K, p-Akt, NF-κB p65, NL-RP3, ASC and caspase-1 after baicalin treatment for 48 h, and ELISA was employed to detect the contents of IL-1 and IL-18 in the supernatents. In order to explore the mechanism of baicalin alleviating the activation of NLRP3 inflammasome, double luciferin and Westen blot analysis were applied to verify the corresponding relationship between let-7i-3p and PIK3CA. RT-qPCR was utilized to determine the expression of let-7i-3p and PI3K before and after baicalin intervention. let-7i-3p interference was used to verify whether baicalin mitigated the activation of enhanced NLRP3 inflammasomes. Results Baicalin (50, 100 mg · L

Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing / 中西医结合学报

OBJECTIVE@#This study tests the efficacy of Bletilla striata polysaccharide (BSP), carboxymethyl chitosan (CMC), baicalin (BA) and silver titanate (ST) in a wound dressings to fight infection, promote healing and provide superior biocompatibility.@*METHODS@#The antibacterial activity of BA and ST was evaluated in vitro using the inhibition zone method. BA/ST/BSP/CMC porous sponge dressings were prepared and characterized. The biocompatibility of BA/ST/BSP/CMC was assessed using the cell counting kit-8 assay. The therapeutic effect of BA/ST/BSP/CMC was further investigated using the dorsal skin burn model in Sprague-Dawley rats.@*RESULTS@#The wound dressing had good antibacterial activity against Escherichia coli and Staphylococcus aureus through BA and ST, while the combination of BSP and CMC played an important role in promoting wound healing. The BA/ST/BSP/CMC porous sponge dressings were prepared using a freeze-drying method with the concentrations of BA and ST at 20 and 0.83 mg/mL, respectively, and the optimal ratio of 5% BSP to 4% CMC was 1:3. The average porosity, water absorption and air permeability of BA/ST/BSP/CMC porous sponge dressings were measured to be 90.43%, 746.1% and 66.60%, respectively. After treatment for 3 and 7 days, the healing rates of the BA/ST/BSP/CMC group and BA/BSP/CMC group were significantly higher than those of the normal saline (NS) group and silver sulfadiazine (SSD) group (P < 0.05). Interleukin-1β expression in the BA/ST/BSP/CMC group at 1 and 3 days was significantly lower than that in the other three groups (P < 0.05). After being treated for 3 days, vascular endothelial growth factor expression in the BA/BSP/CMC group and BA/ST/BSP/CMC group was significantly higher than that in the NS group and SSD group (P < 0.05). Inspection of histological sections showed that the BA/ST/BSP/CMC group and BA/BSP/CMC group began to develop scabbing and peeling of damaged skin after 3 days of treatment, indicating accelerated healing relative to the NS group and SSD group.@*CONCLUSION@#The optimized concentration of BA/ST/BSP/CMC dressing was as follows: 6 mg BSP, 14.4 mg CMC, 0.5 mg ST and 12 mg BA. The BA/ST/BSP/CMC dressing, containing antibacterial constituents, was non-cytotoxic and effective in accelerating the healing of burn wounds, making it a promising candidate for wound healing. Please cite this article as: Gong YR, Zhang C, Xiang X, Wang ZB, Wang YQ, Su YH, Zhang HQ. Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing. J Integr Med. 2023; 21(5): 487-495.

Effect of Baicalin on Pyroptosis of Diffuse Large B-Cell Lymphoma Cell Lines DB and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Baicalin on the proliferation and pyroptosis of diffuse large B-cell lymphoma cell line DB and its mechanism.@*METHODS@#DB cells were treated with baicalin at different concentrations (0, 5, 10, 20, 40 μmol/L). Cell proliferation was detected by CCK-8 assay and half maximal inhibitory concentration (IC50) was calculated. The morphology of pyroptosis was observed under an inverted microscope, the integrity of the cell membrane was verified by LDH content release assay, and the expressions of pyroptosis-related mRNA and protein (NLRP3, GSDMD, GSDME, N-GSDMD, N-GSDME) were detected by real-time fluorescence quantitative PCR and Western blot. In order to further clarify the relationship between baicalin-induced pyroptosis and ROS production in DB cells, DB cells were divided into control group, baicalin group, NAC group and NAC combined with baicalin group. DB cells in the NAC group were pretreated with ROS inhibitor N-acetylcysteine (NAC) 2 mmol/L for 2 h. Baicalin was added to the combined treatment group after pretreatment, and the content of reactive oxygen species (ROS) in the cells was detected by DCFH-DA method after 48 hours of culture.@*RESULTS@#Baicalin inhibited the proliferation of DB cells in a dose-dependent manner (r=-0.99), and the IC50 was 20.56 μmol/L at 48 h. The morphological changes of pyroptosis in DB cells were observed under inverted microscope. Compared with the control group, the release of LDH in the baicalin group was significantly increased (P<0.01), indicating the loss of cell membrane integrity. Baicalin dose-dependently increased the expression levels of NLRP3, N-GSDMD, and N-GSDME mRNA and protein in the pyroptosis pathway (P<0.05). Compared with the control group, the level of ROS in the baicalin group was significantly increased (P<0.05), and the content of ROS in the NAC group was significantly decreased (P<0.05). Compared with the NAC group, the content of ROS in the NAC + baicalin group was increased. Baicalin significantly attenuated the inhibitory effect of NAC on ROS production (P<0.05). Similarly, Western blot results showed that compared with the control group, the expression levels of pyroptosis-related proteins was increased in the baicalin group (P<0.05). NAC inhibited the expression of NLRP3 and reduced the cleavage of N-GSDMD and N-GSDME (P<0.05). Compared with the NAC group, the NAC + baicalin group had significantly increased expression of pyroptosis-related proteins. These results indicate that baicalin can effectively induce pyroptosis in DB cells and reverse the inhibitory effect of NAC on ROS production.@*CONCLUSION@#Baicalin can inhibit the proliferation of DLBCL cell line DB, and its mechanism may be through regulating ROS production to affect the pyroptosis pathway.

Determination of plasma protein binding rate of Shuganning Injection using equilibrium dialysis and UPLC-MS/MS / 中国中药杂志

Traditional Chinese medicine(TCM) compound preparations have complex compositions. As a widely used TCM injection, Shuganning Injection, its in vivo processes are not yet fully understood. Determining the plasma protein binding rate is of great significance for pharmacokinetic and pharmacodynamic studies. In this experiment, the equilibrium dialysis method combined with UPLC-MS/MS technology was used to determine the plasma protein binding rates of 10 components, including p-hydroxyacetophenone, caffeic acid, baicalein, oroxylin A, geniposide, baicalin, cynaroside, oroxylin A-7-O-β-D-glucuronide, scutellarin, and hyperoside, in Shuganning Injection in rat and human plasma to provide a theoretical basis for further elucidating the in vivo processes of Shuganning Injection and guiding clinical medication. The results showed that, except for baicalein and geniposide, the plasma protein binding rates of the other eight components were higher in human plasma than in rat plasma, and there were interspecies differences. In human plasma, except for geniposide, caffeic acid, and baicalin, the plasma protein binding rates of the remaining seven components were above 80%, with baicalein and oroxylin A exceeding 90%. All components exhibit a high level of binding to plasma proteins, with the exception of geniposide.

Baicalin improves inflammatory response of human microglia by regulating cAMP-PKA-NF-κB/CREB pathway / 中国中药杂志

This study aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells, with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway. The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases. First, the safe dosage of BAI was screened, and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h. The cells were assigned into a control group, a model group, and high-, medium-, and low-dose(5, 2.5, and 1.25 μmol·L~(-1), respectively) BAI groups. The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts, as well as the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), and cyclic adenosine monophosphate(cAMP) in the cell supernatant, were measured. Western blot was performed to determine the expression of protein kinase A(PKA), phosphorylated cAMP-response element binding protein(p-CREB), and nuclear factor-kappa B p65(NF-κB p65). Hoechst 33342/PI staining was employed to assess cell apoptosis. High and low doses of BAI were used for treatment in the research on the mechanism. The results revealed that BAI at the concentrations of 10 μmol·L~(-1) and below had no impact on normally cultured HMC3 cells. LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells. However, 5, 2.5, and 1.25 μmol·L~(-1) BAI significantly increased the SOD activity and 5 μmol·L~(-1) BAI significantly decreased the MDA content. In addition, BAI ameliorated the M1 polarization of HMC3 cells induced by LPS, as indicated by cellular morphology. The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α, IL-1β, IL-6, and cAMP in the cell supernatant. Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-regulating the expression of NF-κB p65. Hoechst 33342/PI staining results indicated that BAI mitigated the apoptosis of HMC3 cells. Overall, the results indicated that BAI had protective effects on the HMC3 cells induced by LPS, and could inhi-bit inflammatory response and improve cell apoptosis, which might be related to the regulation of the cAMP-PKA-NF-κB/CREB pathway.

Baicalin Ameliorates Corticosterone-Induced Depression by Promoting Neurodevelopment of Hippocampal via mTOR/GSK3β Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the role of hippocampal neurodevelopment in the antidepressant effect of baicalin.@*METHODS@#Forty male Institute of Cancer Research mice were divided into control, corticosterone (CORT, 40 mg/kg), CORT+baicalin-L (25 mg/kg), CORT+baicalin-H (50 mg/kg), and CORT+fluoxetine (10 mg/kg) groups according to a random number table. An animal model of depression was established by chronic CORT exposure. Behavioral tests were used to assess the reliability of depression model and the antidepressant effect of baicalin. In addition, Nissl staining and immunofluorescence were used to evaluate the effect of baicalin on hippocampal neurodevelopment in mice. The protein and mRNA expression levels of neurodevelopment-related factors were detected by Western blot analysis and real-time polymerase chain reaction, respectively.@*RESULTS@#Baicalin significantly ameliorated the depressive-like behavior of mice resulting from CORT exposure and promoted the development of dentate gyrus in hippocampus, thereby reversing the depressive-like pathological changes in hippocampal neurons caused by CORT neurotoxicity. Moreover, baicalin significantly decreased the protein and mRNA expression levels of glycogen synthase kinase 3β (GSK3β), and upregulated the expression levels of cell cycle protein D1, p-mammalian target of rapamycin (mTOR), doublecortin, and brain-derived neurotrophic factor (all P<0.01). There were no significant differences between baicalin and fluoxetine groups (P>0.05).@*CONCLUSION@#Baicalin can promote the development of hippocampal neurons via mTOR/GSK3β signaling pathway, thus protect mice against CORT-induced neurotoxicity and play an antidepressant role.

Regulation of Baicalin on Growth of Extranodal NK/T Cell Lymphoma Cells through FOXO3/CCL22 Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of baicalin on the growth of extranodal NK/T cell lymphoma (ENKTCL) cells and its related mechanism.@*METHODS@#Normal NK cells and human ENKTCL cells lines SNK-6 and YTS were cultured, then SNK-6 and YTS cells were treated with 5, 10, 20 μmol/L baicalin and set control. Cell proliferation and apoptosis was detected by Edu method and FCM method, respectively, and expressions of BCL-2, Bax, FOXO3 and CCL22 proteins were detected by Western blot. Interference plasmids were designed and synthesized. FOXO3 siRNA interference plasmids and CCL22 pcDNA overexpression plasmids were transfected with PEI transfection reagent. Furthermore, animal models were established for validation.@*RESULTS@#In control group and 5, 10, 20 μmol/L baicalin group, the proliferation rate of SNK-6 cells was (56.17±2.96)%, (51.92±4.63)%, (36.42±1.58)%, and (14.60±2.81)%, respectively, while that of YTS cells was (58.85±2.98)%, (51.38±1.32)%, (34.75±1.09)%, and (15.45±1.10)%, respectively. In control group and 5, 10, 20 μmol/L baicalin group, the apoptosis rate of SNK-6 cells was (5.93±0.74)%, (11.78±0.34)%, (28.46±0.44)%, and (32.40±0.37)%, respectively, while that of YTS cells was (7.93±0.69)%, (16.29±1.35)%, (33.91±1.56)%, and (36.27±1.06)%, respectively. Compared with control group, the expression of BCL-2 protein both in SNK-6 and YTS cells decreased significantly (P<0.001), and the expression of Bax protein increased in SNK-6 cells only when the concentration of baicalin was 20 μmol/L (P<0.001), while that in YTS cells increased in all three concentrations(5, 10, 20 μmol/L) of baicalin (P<0.001). The expression of FOXO3 protein decreased while CCL22 protein increased in ENKTCL cell lines compared with human NK cells (P<0.001), but the expression of FOXO3 protein increased (P<0.01) and CCL22 protein decreased after baicalin treatment (P<0.001). Animal experiments showed that baicalin treatment could inhibit tumor growth. The expression of CCL22 protein in ENKTCL tissue of nude mice treated with baicalin decreased compared with control group (P<0.01), while the FOXO3 protein increased (P<0.05). In addition, FOXO3 silencing resulted in the decrease of FOXO3 protein expression and increase of CCL22 protein expression (P<0.01, P<0.001).@*CONCLUSION@#Baicalin can inhibit proliferation and promote apoptosis of ENKTCL cell lines SNK-6 and YTS, up-regulate the expression of Bax protein, down-regulate the expression of BCL-2 protein, and down-regulate the expression of CCL22 protein mediated by FOXO3. Animal experiment shown that the baicalin can inhibit tumor growth. Baicalin can inhibit the growth and induce apoptosis of ENKTCL cells through FOXO3/CCL22 signaling pathway.

Effect and mechanism of baicalin on the proliferation and migration of human periodontal ligament stem cells induced by lipopolysaccharide / 中国药房

OBJECTIVE To explore the regulatory effects of baicalin on the proliferation and migration of human periodontal ligament stem cells （hPDLSCs） induced by lipopolysaccharide （LPS） and Janus protein tyrosine kinase 2 （JAK2）/signal transduction and transcription activator 3 （STAT3） signaling pathways. METHODS hPDLSCs were divided into control group， LPS group， different concentration baicalin groups （0.1， 1 and 10 mg/L）. ELISA method and CCK-8 assay were used to determine the contents of cell inflammatory factors [interleukin 6 （IL-6）， IL-1β and tumor necrosis factor-α （TNF-α）] and cell viability， so as to screen the optimal concentration of baicalin for follow-up pathway validation experiments. The cells were then divided into control group， LPS group， optimal baicalin concentration group and inhibitor group （10 μg/mL LPS+1 mg/L baicalin +3 μmol/L JAK2/STAT3 pathway inhibitor AG490）. After treated for 24 h， the proliferation rate of hPDLSCs， apoptosis rate， migration rate， invasion cell number， mRNA and protein expressions of Cyclin D1 and caspase-3， the expression of JAK2/STAT3 pathway-related proteins were all detected. RESULTS According to cell inflammatory factors and cell viability， 1 mg/L was selected as the optimal concentration of baicalin. Compared with control group， cell proliferation rate， migration rate， invasion cell number， Cyclin D1 mRNA and protein expression were significantly decreased in LPS group， while cell apoptosis rate， caspase-3 mRNA and protein expression， p-JAK2 and p-STAT3 protein expression were significantly increased （P＜0.05）. After treated with 1 mg/L baicalin， the above indexes were reversed significantly （P＜0.05）. The improvement of above indexes in the inhibitor group was more obvious （P＜0.05）. CONCLUSIONS Baicalin can promote the proliferation， migration and invasion of LPS-induced hPDLSCs and inhibit their apoptosis and inflammation by blocking the JAK2/STAT3 pathway.

Role of Akt/GSK3β signal pathway in baicalin improving the cognitive function of mice with brain injury induced by mechanical ventilation / 中华行为医学与脑科学杂志

Objective:To investigate the effect of baicalin on cognitive function of mice with brain injury induced by mechanical ventilation and its mechanism.Methods:Seventy two C57BL6 mice, weighing 20-25 g, aged 8-12 weeks, were randomly divided into control group (group C), mechanical ventilation group (group V), baicalin group (group B), baicalin+ Akt inhibitor MK-2206 group (group BM) according to random number table method, with 18 in each group.Mice in group C did not have mechanical ventilation and breathed air independently for 6 hours.Mice in group V received mechanical ventilation for 6 hours.Mice in group B and group BM were intraperitoneally injected with baicalin 100 mg/kg 30 minutes before mechanical ventilation, and mice in group BM were injected intraventricular with Akt inhibitor MK-2206 300 μg/kg 60 minutes before mechanical ventilation.Six mice in each group were randomly selected to test their learning and memory abilities by Morris water maze test 1st day before mechanical ventilation and 3rd day and 7th day after mechanical ventilation.One day after mechanical ventilation, six mice in each group were killed, and the brain tissue was taken.TUNEL method was used to detect the neuronal apoptosis in hippocampal CA1 area, and the apoptosis index was calculated.One day after mechanical ventilation, six mice in each group were killed, and the hippocampus was taken, Western blot was used to detect the protein expressions of caspase-3, caspase-9, Akt, p-Akt, GSK-3β and p-GSK-3β.SPSS 22.0 software was used for statistical analysis of data, repeated measure ANOVA and one-way ANOVA were used for comparison between multiple groups.LSD- t test was used for further pairwise comparison. Results:The results of water maze test showed that the time and group interaction of the four groups were not significant ( F=1.14, P>0.05), the main effect of time and group were both significant ( F=47.36, 59.65, both P<0.05). At 3rd day and 7th day after mechanical ventilation, the escape latencies of mice in group V were higher than those in group C (both P<0.05), and the numbers of platform crossing were lower than those in group C (both P<0.05). And 3 days and 7 days after mechanical ventilation, the escape latencies of mice in group B were lower than those in group V (both P<0.05) and the numbers of platform crossing were higher than those in group V (both P<0.05). The escape latenies of mice in BM group on the 3rd and 7th day were higher than those in group B (both P<0.05), and the numbers of platform crossing were lower than those in group B on the 3rd day and 7th day after mechanical ventilation(both P<0.05). TUNEL and Western blot results showed that apoptosis index of hippocampal neurons and expression levels of apoptosis-related proteins caspase-3 and caspase-9 were significant different in the four groups ( F=51.42, 41.21, 40.19, all P<0.05). The apoptosis index of hippocampal neurons ((40.6±3.9)%), the expression levels of caspase-3 (4.93±0.92) and caspase-9 (4.81±0.88) in the hippocampus of mice in group V were higher than those in group C ((13.7±1.4)%, (1.87±0.27), (1.71±0.25), all P<0.05), the apoptosis index of hippocampal neurons ((15.6±1.6)%), the expression levels of caspase-3 (1.95±0.30) and caspase-9 (1.76±0.28) in group B were lower than those in group V ((40.6±3.9)%, (4.93±0.92), (4.81±0.88), all P<0.05), the apoptosis index of hippocampal neurons ((27.8±2.7)%), the expression levels of caspase-3 (3.58±0.61) and caspase-9 (3.49±0.57) in BM group were higher than those in group B ((15.6±1.6)%, (1.95±0.30), (1.76±0.28), all P<0.05). Expression level of p-Akt, p-GSK-3β in hippocampal tissues of the four group of mice were significantly different ( F=37.54, 43.23, both P<0.05). The expression level of p-Akt (0.51±0.06) and p-GSK-3β (0.47±0.05) of hippocampal tissues of mice in group V were lower than those of group C ((1.07±0.10), (1.11±0.12), both P<0.05), the expression level of p-Akt (0.99±0.10) and p-GSK-3β (1.08±0.09) of hippocampal tissues of mice in group B were higher than those of group V (both P<0.05), the expression level of p-Akt (0.83±0.08) and p-GSK-3β (0.81±0.07) of hippocampal tissues of mice in group BM were lower than those in group B (both P<0.05). Conclusion:Baicalin can improve the cognitive function of mice with brain injury induced by mechanical ventilation, which is related with activation of Akt/GSK-3β signaling pathway and inhibition of hippocampal neuron apoptosis.

Optimization of preparation technology of baicalin lipid nano foam aerosol / 中国药房

OBJECTIVE To optimize the pr eparation technology of the baicalin lipid nano foam aerosol （BC-LN-FA）. METHODS Baicalin lipid nanoparticle （BC-LN）and BC-LN-FA were prepared by the thin film dispersion method and homogeneous emulsification method ，respectively，using baicalin （BC） as the model drug. The preparation technology was optimized by Box-Behnken design-response surface methodology using particle size and encapsulation efficiency （EE）as indexes ，with dosage ， emulsifier dosage ，co-emulsifier dosage and homogenization time as factors. The morphology ，particle size ，polymerdispersity index（PDI），EE，the viscosity ，the foam dissolution rate and in vitro transdermal release of BC-LN-FA were characterized. RESULTS The optimal technology included 25 mg BC ，40 mg emulsifier （mass ratio of stearic acid-soybean lecithin-glycerol was 1∶1∶1），30 mg co-emulsifier （mass ratio of octadecanol-lactic acid was 1∶1），homogenization time of 20 min. Results of 3 times of validation tests showed that particle size of prepared BC-LN-FA was （151.70±2.40）nm，EE was （68.62±1.16）％；the deviation of them from the predicted value （particle size of 150.80 nm，EE of 67.02％）were 0.60％ and 2.39％ respectively. The BC-LN-FA prepared by the optimal process was light yellow opalescence ，uniform in particle size and round-like in shape. The viscosity，the foam dissolution rate ，the content of BC and PDI were （122.92±5.09）mPa·s，（65.32±3.22）％，（7.01±0.12）％ and（0.199±0.006），respectively. At 48 h，the cumulative release rates of BC-LN-FA in phosphate buffer saline （PBS）at pH 7.4， 6.8，5.0 were（54.12±2.69）％，（57.85±4.25）％ and（59.47±1.83）％，respectively；those of free BC in PBS at pH 7.4 was only （15.04±1.43）％. CONCLUSIONS The optimized technology is stable and feasible. Prepared BC-LN-FA has a uniform particle size，high digestion rate and certain viscosity.

Baicalin inhibits LPS/IFN-γ-induced inflammation via TREM2/TLR4/NF-κB pathway in BV2 cells / 中国中药杂志

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferon γ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB) pathway. Specifically, LPS and IFN-γ were used to induce inflammation in mouse microglia BV2 cells. Then the normal group, model group, low-dose(5 μmol·L~(-1)) baicalin group, medium-dose(10 μmol·L~(-1)) baicalin group, high-dose(20 μmol·L~(-1)) baicalin group, and minocycline(10 μmol·L~(-1)) group were designed. Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field. The expression of interleukin-1β(IL-1β), interleukin-4(IL-4), inducible nitric oxide synthase(iNOS), interleukin-6(IL-6), interleukin-10(IL-10), and arginase-1(Arg-1) mRNA was detected by real-time quantitative PCR, the protein expression of tumor necrosis factor-α(TNF-α), IL-1β, TREM2, TLR4, inhibitor kappaB-alpha(IκBα), p-IκBα, NF-κB p65 and p-NF-κB p65 by Western blot, and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence. Compared with the normal group, most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology, and the model group showed significant increase in the mRNA levels of IL-1β, IL-6, and iNOS, decrease in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), rise of the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.01), reduction in TREM2 protein expression, and increase in the expression of NF-κB p65 in nucleus. Compared with the model group, baicalin groups and minocycline group showed the recovery of BV2 cell morphology, significant decrease in the mRNA levels of IL-1β, IL-6 and iNOS, increase in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), reduction in the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.05), rise of TREM2 protein expression, and decrease in the expression of NF-κB p65 in nucleus. In summary, these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB, thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.

Baicalin treats cerebral ischemia reperfusion-induced brain edema in rats by inhibiting TRPV4 and AQP4 of astrocytes / 中国中药杂志

This study aims to explore the pharmacodynamic effect of baicalin on rat brain edema induced by cerebral ischemia reperfusion injury and discuss the mechanism from the perspective of inhibiting astrocyte swelling, which is expected to serve as a refe-rence for the treatment of cerebral ischemia with Chinese medicine. To be specific, middle cerebral artery occlusion(suture method) was used to induce cerebral ischemia in rats. Rats were randomized into normal group, model group, high-dose baicalin(20 mg·kg~(-1)) group, and low-dose baicalin(10 mg·kg~(-1)) group. The neurobehavior, brain index, brain water content, and cerebral infarction area of rats were measured 6 h and 24 h after cerebral ischemia. Brain slices were stained with hematoxylin and eosin(HE) for the observation of pathological morphology of cerebral cortex after baicalin treatment. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of total L-glutathione(GSH) and glutamic acid(Glu) in brain tissue, Western blot to measure the content of glial fibrillary acidic protein(GFAP), aquaporin-4(AQP4), and transient receptor potential vanilloid type 4(TRPV4), and immunohistochemical staining to observe the expression of GFAP. The low-dose baicalin was used for exploring the mechanism. The experimental results showed that the neurobehavioral scores(6 h and 24 h of cerebral ischemia), brain water content, and cerebral infarction area of the model group were increased, and both high-dose and low-dose baicalin can lower the above three indexes. The content of GSH dropped but the content of Glu raised in brain tissue of rats in the model group. Low-dose baicalin can elevate the content of GSH and lower the content of Glu. According to the immunohistochemical staining result, the model group demonstrated the increase in GFAP expression, and swelling and proliferation of astrocytes, and the low-dose baicalin can significantly improve this situation. The results of Western blot showed that the expression of GFAP, TRPV4, and AQP4 in the cerebral cortex of the model group increased, and the low-dose baicalin reduce their expression. The cerebral cortex of rats in the model group was severely damaged, and the low-dose baicalin can significantly alleviate the damage. The above results indicate that baicalin can effectively relieve the brain edema caused by cerebral ischemia reperfusion injury in rats, possibly by suppressing astrocyte swelling and TRPV4 and AQP4.

Baicalin Alleviates LPS-Induced Acute Lung Injury in Rats Through p38 MAPK/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects and mechanism of baicalin （BA） on lipopolysaccharide （LPS）-induced acute lung injury in rats. MethodEighty healthy male SD rats were randomly divided into the control group， model group， low-dose BA （BA-L） group， medium-dose BA （BA-M） group， high-dose BA （BA-H） group， dexamethasone （DEX） group， SB203580 group， and BA + SB203580 group， with 10 rats in each group. The rats in the BA-L， BA-M， and BA-H groups were injected intraperitoneally with different doses （10， 50， 100 mg·kg-1） of BA solution， the ones in the DEX group with 5 mg·kg-1 DEX solution， the ones in the SB203580 group with 0.5 mg·kg-1 SB203580 solution， the ones in the BA + SB203580 group with 100 mg·kg-1 BA solution and 0.5 mg·kg-1 SB203580， and those in both the control group and model group with the same volume of normal saline， once per day， for seven successive days. One hour after the last administration， rats in all groups except for the control group were given 5 mg·kg-1 LPS via intratracheal instillation for inducing the acute lung injury， whereas those in the control group received the same volume of normal saline solution. Twelve hours later， the lung tissues were sampled and stained with htoxylin-eosin （HE） for observing the pathological changes， followed by the counting of the total number of cells and neutrophils in bronchoalveolar lavage fluid （BALF）. The wet/dry weight ratio of the lung tissue and the contents of serum superoxide dismutase （SOD） and malondialdehyde （MDA） were measured. The activity of reactive oxygen species （ROS） in the lung tissue was detected by immunofluorescence and the levels of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in BALF by enzyme-linked immunosorbent assay （ELISA）. Immunohistochemistry （IHC） was conducted to determine the relative expression of p-p38 mitogen-activated protein kinase （MAPK） and Western blotting was carried out to detect the protein expression levels of p-p38 MAPK， thioredoxin interacting protein （TXNIP）， NOD-like receptor protein 3 （NLRP3）， and cysteinyl aspartate specific protease-1 （Caspase-1） in the lung tissue. ResultCompared with the control group， the model group displayed inflammatory pathological changes in lung tissue， elevated wet/dry weight ratio， total number of cells and neutrophils in BALF， and ROS and MDA levels （P<0.01）， decreased SOD activity （P<0.01）， and up-regulated IL-1， IL-18， IL-6， TNF-α， p-p38 MAPK， NLRP3， and Caspase-1 expression （P<0.01）. Compared with the model group， BA at different doses， SB203580， and BA + SB203580 all effectively alleviated the pathological changes in lung tissue induced by LPS， reduce the lung wet/dry weight ratio， the total number of cells and neutrophils in BALF， and ROS and MDA levels （P<0.05，P<0.01）， enhanced the activity of SOD （P<0.05，P<0.01）， and down-regulated the expression of IL-1β， IL-18， IL-6，TNF-α， p-p38 MAPK， NLRP3， and Caspase-1 in lung tissue （P<0.05，P<0.01）. ConclusionBA has a protective effect against LPS-induced acute lung injury， which may be related to its inhibition of p38MAPK/NLRP3 signaling pathway and the improvement of inflammatory response.