Baitouweng Tang Suppresses Colorectal Cancer HCT116 Cells by Regulating Hedgehog Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect of Baitouweng Tang （BTWT） on the apoptosis of human colorectal cancer HCT116 cells and decipher the underlying mechanism based on the Hedgehog （Hh） signaling pathway. MethodHCT116 cells were treated with BTWT （25， 50， 100， 200， 500， 750， and 1 000 mg·L-1） for 24 h， and then the cell proliferation was detected by methyl thiazolyl tetrazolium （MTT） colorimetry. Five groups were designed for the treatment of HCT116 cells， including a blank control group， BTWT groups （125， 250， and 500 mg·L-1）， and a positive control （5-fluorouracil， 5-FU， 40 mmol·L-1） group. The cell morphology was observed under an inverted microscope. The migration of the cells was detected by scratch test， and the apoptosis by Hoechest 33324/propidium iodide （PI） staining and flow cytometry. Western blot was employed to determine the protein levels of sonic hedgehog （SHh）， GLI family zinc finger protein 1 （Gli1）， smoothened （Smo）， suppressor of fused （SuFu）， cellular-myelocytomatosis viral oncogene （c-Myc）， and the apoptosis-related proteins B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. The quantitative real-time reverse transcription PCR （Real-time PCR） was employed to determine the mRNA levels of Bax， Bcl-2， SHh， Gli1， Smo， SuFu， and c-Myc. ResultCompared with the blank control group， BTWT changed the cell morphology （making the cell become round with dense nucleus）， inhibited the proliferation of HCT116 cells in a dose-dependent manner， decreased the ability of migration （P<0.05， P<0.01）， and increased apoptotic cells. Compared with the blank control group， BTWT （500 mg·L-1） treatment for 24 h up-regulated the protein and mRNA levels of Bax （P<0.05， P<0.01） and down-regulated the protein and mRNA levels of Bcl-2 in HCT116 cells （P<0.05， P<0.01）. Moreover， the treatment down-regulated the mRNA and protein levels of SHh， Gli1， Smo， and c-Myc （P<0.05， P<0.01） and up-regulated the mRNA and protein levels of SuFu （P<0.05， P<0.01）. ConclusionBTWT inhibited the proliferation and migration and induced the apoptosis of colorectal cancer HCT116 cells by down-regulating the Hh signaling pathway.

Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Qiling Baitouweng Tang （QLBTWT） on proliferation and apoptosis， Janus kinase 2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） signaling pathway and interleukin-10 （IL-10） in diffuse large B-cell lymphoma （DLBCL）. MethodWith human DLBCL cells OCI-LY10 and U2932 as research objects， cell proliferation was detected by cell counting kit-8 （CCK-8） assay. After treatment with 0， 4.6， 9.3， 18.7， 37.5， 75， 150 mg·L-1 QLBTWT for 24 h， the half-inhibitory concentration （IC50） of OCL-LY10 and U2932 cells was calculated to be 9.33， 16.13 mg·L-1， respectively， based on which， 9.5， 19， 38 mg·L-1 QLBTWT were selected for subsequent experiments. After 0， 9.5， 19， 38 mg·L-1 QLBTWT treatment for 24 h， the zymogen activities of Caspase-3， Caspase-8 and Caspase-9 in OCI-LY10 and U2932 cells were detected using corresponding activity assay kits （colorimetric）， and the IL-10 expression was detected by enzyme-linked immuno sorbent assay （ELISA）. The apoptosis rate and cell cycle of OCI-LY10 and U2932 cells treated with different concentrations of QLBTWT for 24 h were detected by flow cytometry. The expressions of apoptosis-related proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly adenosine diphosphate ribose polymerase （cleaved PARP）， cleaved Caspase-3］， JAK2， STAT3， phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3） pathway proteins， and c-Myc protein in OCL-LY10 and U2932 cells after 24 h treatment with 0， 9.5， 19， 38 mg·L-1 QLBTWT were all tested by Western blot. ResultAfter QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， cell proliferation was inhibited in each QLBTWT group compared with that in the control group （P<0.05， P<0.01）. The zymogens of Caspase-3， Caspase-8 and Caspase-9 were activated （P<0.01）， and there was an increase in cell apoptosis （P<0.05， P<0.01） and cell cycle arrest at Gap phase1 （G1） phase in 9.5， 19 and 38 mg·L-1 QLBTWT group （P<0.05， P<0.01）. After 9.5， 19 and 38 mg·L-1 QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， the expressions of Bcl-2， p-JAK2 and p-STAT3 proteins were decreased （P<0.01）， and the expressions of Bax， cleaved PARP and cleaved Caspase-3 proteins were increased （P<0.01）， but no significant change was observed in the expressions of JAK2 and STAT3 proteins. Compared with the conditions in the control group， the expressions of c-Myc， p-JAK2， and p-STAT3 proteins were down-regulated in 19 mg·L-1 QLBTWT group and 19 mg·L-1 QLBTWT+10 μg·L-1 IL-10 group （P<0.05， P<0.01）， and up-regulated in 10 μg·L-1 IL-10 group （P<0.05， P<0.01）， while there was no difference in JAK2/STAT3 proteins. ConclusionQLBTWT can inhibit proliferation and induce apoptosis of human DLBCL cells OCI-LY10 and U2932， and the potential mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

Effect of Sishenwan， Baitouweng Tang， and Lianlitang on Ulcerative Colitis in Rats / 中国实验方剂学杂志

Objective:To explore the mechanism of Sishenwan， Baitouweng Tang， and Lianlitang in the treatment of ulcerative colitis （UC）， and compare their efficacies on UC in rats. Method:Ninety SD rats of SPF grade were randomly divided into blank group （distilled water， 2 mL·d^-1） and experimental group. The rats in the experimental groups were administered with 2，4，6-trinitrobenzene sulfonic acid （TNBS） by clysis to induce the UC model. Subsequently， the model rats were divided into a model group （distilled water， 2 mL·d^-1）， positive group ［sulfasalazine （SASP）， 0.4 g·kg^-1·d^-1］， Sishenwan group （1.76 g·kg^-1·d^-1）， a Baitouweng Tang group （1.40 g·kg^-1·d^-1）， and Lianlitang group （2.13 g·kg^-1·d^-1） according to the random number table. The rats in each group were dosed at 2 mL·d^-1 for 14 days. The pathological score for colonic mucosa was detected. Cytokines were detected by the cytokine chip. The enzyme-linked immunosorbent assay （ELISA） was used to detect the free triiodothyronine （FT₃）， glucagon-like peptide-1 （GLP-1）， and corticosterone （CORT） in plasma， and neurotensin （NT）， substance P （SP）， vasoactive intestinal peptide （VIP）， and somatostatin （SST） in colon tissues. Result:Compared with the normal group， the model group showed increased colon mass-length ratio and pathological score for colonic mucosa （<italic>P</italic><0.01）， infiltration of massive lymphocytes， disordered or absent intestinal villi， elevated levels of cytokine-induced neutrophil chemoattractant-1/2<italic>α</italic>/<italic>β</italic>/3 （CINC-1/2<italic>α</italic>/<italic>β</italic>/3）， interleukin-1<italic>α</italic> （IL-1<italic>α</italic>）， interferon-inducible protein-10 （IP-10） and other factors in colon tissues （<italic>P</italic><0.05）， dwindled CORT and GLP-1 levels in plasma （<italic>P</italic><0.05）， and increased SP content in colon tissues （<italic>P</italic><0.05）. Compared with the results in the model group， the mucosal injury in the colon of rats in each drug group was relieved. The levels of IL-1<italic>α</italic>， IP-10， lipopolysaccharide-inducible CXC chemokine （LIX）， and L-selectin of rats in the Lianlitang group and Sishenwan group were reduced （<italic>P</italic><0.05）， and the CINC-3 and IL-17 levels were diminished in the Baitouweng Tang group （<italic>P</italic><0.05）. The levels of CINC-1/3， IL-1<italic>α</italic>， and IP-10 were reduced in the SASP group （<italic>P</italic><0.05）. The plasma FT₃ was up-regulated in the Lianlitang group， and the plasma GLP-1 levels were elevated in the three Chinese medicine groups （<italic>P</italic><0.05）. The VIP content in colon tissues of the Sishenwan group and Baitouweng Tang group was down-regulated （<italic>P</italic><0.05）， and the SST content in colon tissues of the SASP group was significantly up-regulated （<italic>P</italic><0.01）. Conclusion:The intervention of Lianlitang and Sishenwan on UC was significant， and the underlying mechanism of action might be related to inflammation inhibition and immune balance by regulating the cytokine network. The efficacy of Lianlitang was predominant， followed by Sishenwan and Baitouweng Tang.

Jiawei Baitouweng Decoction affects intestinal mucosal tight junction proteins in rats with ulcerative colitis through p38 MAPK-MLCK signaling pathway / 中国中药杂志

The aim of this paper was to explore the effect and mechanism of Jiawei Baitouweng Decoction(JWBTW) against ulcerative colitis(UC) from the perspective of intestinal mucosal tight junction proteins. From 60 SPF-grade male SD rats, 10 were randomly selected as the blank control, and the remaining 50 were treated with 3% dextran sodium sulfate(DSS) solution to induce UC and then randomized into the model group, mesalazine group, and low-, medium-, and high-dose JWBTW( L-JWBTW, M-JWBTW and H-JWBTW) groups, with 10 rats in each group. After successive medication for 14 days, the rat general conditions like body weight and stool were observed and the disease activity index(DAI) was calculated. The pathological changes in colon tissue was observed under a microscope for injury severity scoring and histopathological scoring. The serum endotoxin content was determined by limulus assay, followed by the measurement of protein expression levels of ZO-1, occludin, claudin-1, p38 MAPK, MLCK, MLC2 and p-MLC in colon tissue by Western blot. The results showed that compared with the blank group, the model group exhibited significantly reduced body weight, elevated DAI, injury severity and histopathological scores and serum endotoxin content, up-regulated protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and down-regulated ZO-1, occludin and claudin-1. Compared with the model group,mesalazine and JWBTW at each dose obviously increased the body weight, lowered the DAI, injury severity and histopathological scores and serum endotoxin content, down-regulated the protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and up-regulated the ZO-1, occludin and claudin-1, with the most obvious changes noticed in the H-JWBTW group. All these have indicated that JWBTW exerts the therapeutic effect against UC by inhibiting the activation of p38 MAPK/MLCK pathway, reversing the protein expression levels of occludin, claudin-1 and ZO-1, decreasing the serum endotoxin content, promoting the repair of intestinal mucosal mechanical barrier, maintaining the integrity of tight junctions, and reducing the permeability of intestinal mucosa.

Efficacy and Immune Mechanism of Baitouweng Tang on Ulcerative Colitis in Rats / 中国实验方剂学杂志

Objective: To investigate the therapeutic effect and immune mechanism of Baitouweng Tang on ulcerative colitis(UC) rats. Method: The mode of UC rats was made of 2, 4-dinitrochlorobenzene (DNCB)/ethanol enema. Rats were randomly divided into control group, model group, mesalazine group, and high-dose, middle-dose and low-dose Baitouweng Tang groups. The mesalazine group were administered with mesalazine (0.5 g·kg-1). Baitouweng Tang groups were given Baitouweng Tang (10, 5, 2.5 g·kg-1), while the other groups were given double steaming water. After 7 days of continuous administration, the general condition and disease activity index of rats in each group were observed. After anesthesia in rats, blood was taken from the abdominal aorta. Then the rats were put to death, and the length and morphological observation of the colon were measured. Ultraviolet spectrophotometry detection was used to detect the activities of myeloperoxidase (MPO) in blood and colon tissue. The levels of P-selectin, macrophage migration inhibitory factor (MIF) and thromboxane B2 (TXB2) in blood and colon tissues were detected by enzyme-linked immunosorbent assay(ELISA). Immunohistochemistry and Western blot methods were undertaken to determine the expressions of Toll-like receptor 4 (TLR4) and nuclear transcription factor-κB (NF-κB) proteins in colon tissue. Result: Compared with the model group, the rats in model group showed severe symptoms, such as loose stools, diarrhea and bloody stools, while Baitouweng Tang obviously ameliorated them. Moreover, Baitouweng Tang significantly reduced DAI, colon general and pathological scores, which were high in model group(P2 in the serum and colon tissues of the model group were obviously increased(PκB in colons of model group were markedly higher than those in control group(PPConclusion: Baitouweng Tang could inhibit TLR4/NF-κB signaling pathway in treatment of ulcerative colitis, and reduce the expressions of P-selectin, MPO, MIF and TXB2, and thus promoting intestinal mucosal repair and improving intestinal function.