Effects of Zuogui Pill (左归丸) on Wnt Singal Transduction in Rats with Glucocorticoid-induced Osteoporosis

Objective: To reveal the mechanism of Zuogui Pill (左归丸) in treatment of glucocorticoid-induced osteoporosis from the angle of the Wnt signal transduction pathway and to provide further experimental evidence for expounding the scientific connotation of "the kidney dominating the bones" in TCM. Methods: Forty-two male Wistar rats were selected and randomly divided into three groups, control group (n=12), model group (n=15) and Zuogui Pill group (n=15). Form the beginning, The rats were injected dexamethasone for eight weeks to make the model of osteoporosis, and the Zuogui Pill were administered intragastrically to the rats of Zuogui Pill group for eight weeks. The relative morphological parameters were measured in the undecalcified tibial slices. And the protein expression levels of Wnt1, LRP-5 and β-catenin in rat tibial osteoblasts (OB) and bone marrow stromal cells (BMC) were detected by immunohistochemistry. Results: Compared with the control group, TBV% and TFS% decreased significantly, while TRS% increased significantly, and the protein expression of Wnt1, LRP-5 and β-catenin in OB and BMC decreased significantly in the model group. And compared with the model group, TBV% and TFS% increased significantly, and expression levels of Wnt1, LRP-5 and β-catenin proteins increased significantly in the Zuogui pill group. Conclusion: Zuogui Pill can prevent and treat glucocorticoid-induced osteoporosis in rats by up-regulating the expression of the key signal molecules Wnt1, LRP-5 and β-catenin in Wnt signal transduction pathway.     

Effect of Jinguo Weikang Capsule （金果胃康胶囊） on Proto-oncogene Expression of Gastric Mucosa in Rats with Gastric Precancerous Lesions

Objective： To study the effect of Jinguo Weikang Capsule （金果暖康胶囊, JWC） on the gene expression of H-ras, epidermal growth factor receptor （EGFR）, P53 and C-myc of the gastric mucosa in rats with gastric precancerous lesions, and to investigate the action mechanism of JWC on gastric precancerous lesions. Methods： A rat model with paratypical proliferation of the gastric epithelium mucosa was established by using ^60Co irradiation. Rats were divided into the normal group, model group, high-, medium-, low-dose JWC treatment groups, and the vitacoenzyme control group, and were treated for 30 days. The expression of H-ras, EGFR, P53 and C-myc genes of the gastric mucosa was detected by using immunohistochemical methods. Results： The expression and over-expression rates of H-ras, EGFR, P53 and C-myc gene in the high- and medium-dose JWC treatment groups were significantly lower （P〈0.05） as compared with those of the model group. Conclusion： JWC can inhibit the expression of the H-ras, EGFR, P53 and C-myc genes expression of the gastric mucosa in rats, which may be one of mechanisms involved in suppressing or reversing gastric carcinogenesis.     

PIN1 Gene Overexpression and β-catenin Gene Mutation/Expression in Hepatocellular Carcinoma and Their Significance

The evolution of hepatocellular carcinoma (HCC) is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl- isomerase PIN1 gene, the mutation in exon 3 of β-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mu-tations in exon 3 of β-catenin gene and differential expression of β-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The re-sults indicated: (1) 44.8% (13/29) cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues (χ2 =32.63, P<0.05), especially in cytoplasm and nucleus, while there was lower level of PIN1 expression in non-cancerous tissues; (2) 58.6% (17/29) HCC tissues showed β-catenin protein accumulation in cytoplasm and nucleus. 46.2% (6/13) HCC tissues indicated β-catenin protein accumulation with higher level of PIN1 expression, while 53.8% (7/13) HCC tis-sues indicated β-catenin protein accumulation with lower level or trace of PIN1 expression (χ2 =0.00, P>0.05); (3) 24.1% (7/29) of primary tumor lesions carried gene mutations in exon 3 of β-catenin, and accompanied by β-catenin protein accumulation. There was no mutation in non-cancerous tissues. All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of β-catenin gene in tissues with high PIN1 expression level (χ2=58.12, P<0.05). So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way dif-ferent from β- catenin gene mutation.     

Gastric cancer induced by N—methyl—N‘—nitro—N—nitrosoguanidine in rat with ulcers and expressions of ras and c—erbB2 genes

Objective:To observe the series of pathological changes during the development of gastric adenocarcinoma in ulcerative rats induced by N-methyl-N‘-nitrosoguanidine(MNNG),and the expression profile of related oncogenic protein.Methods:MNNG was administered in rats with ulcers due to acetic acid treatment to induce gastric cancer,and the protein expressions of ras and c-erbB2 genes in the ulcer were examined immunohistochemically along with pathological examination.Results:The incidence of gastric adenocarcinoma in the model group reaches 40%(6/15),while none of the rats developed cancer in the control group with ulcers.Postiive expressions of the proteins of p21 ras and c-erbB2 were observed in the tissues undergoing canceration in the 6 rats of model group,but were not observed in the 5 control rats;p53 protein expression,however,failed to be detected in both groups.Conclusion:A new animal model of gastric cancer has been established in rats with gastric ulcer after MNNG treatment,which may facilitate the pharmacological reserach of gastric cancer.     

Positive Effects of Qing'e Pill(青娥丸)on Trabecular Microarchitecture and its Mechanical Properties in Osteopenic Ovariectomised Mice

Objective: To investigate the impact of Qing'e Pill(青娥丸,QEP) on the cancellous bone microstructure and its effect on the level of β-catenin in a mouse model of postmenopausal osteoporosis.Methods: Ninety-six 8-week-old speci?c pathogen free C57 BL/6 mice were randomly divided into 4 groups(24/group): sham,ovariectomised osteoporosis model,oestradiol-treated,and QEP-treated groups.Three months after surgery,the third lumbar vertebra and left femur of the animals were dissected and scanned using micro-computed tomography(micro-CT) to acquire three-dimensional(3 D) parameters of their cancellous bone microstructure.The impact of ovariectomy,the effect of oestradiol and QEP intervention on cancellous bone microstructure,and the expression of β-catenin were evaluated.Results: The oestradioland the QEP-treated groups exhibited a signi?cant increase in the bone volume fraction,trabecular number,trabecular thickness,bone surface to bone volume ratio(BS/BV),and β-catenin expression compared with those of the model group(P0.05).In contrast,the structure model index,trabecular separation,and BS/BV were signi?cantly decreased compared with those of the ovariectomised osteoporosis model group(P0.05).No differences were observed in the above parameters between animals of the QEP-and oestradiol-treated groups.Conclusions: The increased β-catenin expression may be the mechanism underlying QEP's improvement of the cancellous bone microstructure in ovariectomised mice.Our ?ndings provide a scienti?c rationale for using QEP as a dietary supplement to prevent bone loss in postmenopausal women.     

Association of Serum Dkk-1 Levels with β-catenin in Patients with Postmenopausal Osteoporosis

Wnt signaling plays an important role in the bone development and remodeling. The Wnt antagonist Dkk-1 is a potent inhibitor of bone formation. The aims of this study were firstly to compare the serum Dkk-1 levels in postmenopausal osteoporosis patients with age-matched healthy controls, and secondly, to assess the possible relationship between Dkk-1 and β-catenin, sclerostin, or bone turnover markers [CTX, PINP, N-MID-OT and 25(OH)D] in the setting of postmenopausal osteoporosis. A total of 350 patients with postmenopausal osteoporosis and 150 age-matched healthy controls were enrolled, and the serum levels of Dkk-1, β-catenin, sclerostin, OPG, and RANKL were detected by ELISA, and bone turnover markers [CTX, PINP, N-MID-OT and 25(OH)D] were measured by Roche electrochemiluminescence system in two groups. Serum Dkk-1 levels were significantly higher in postmenopausal osteoporosis group than in control group(P<0.001). Univariate analyses revealed that serum Dkk-1 levels were weakly negatively correlated to β-catenin(r=–0.161, P=0.003) and OPG(r=–0.106, P=0.047), while multiple regression analysis showed a negative correlation between serum Dkk-1 levels with β-catenin(β=–0.165, P=0.009) and BMD(β=–0.139, P=0.027), and a positive correlation between serum Dkk-1 levels and CTX(β=0.122, P=0.040) in postmenopausal osteoporosis group. No similar correlations ware observed in control group. The results provided evidence for the role of Dkk-1 in bone metabolism and demonstrated the link of Dkk-1 and Wnt/β-catenin in some ways.     

Implication of Receptor Activator of NF-κB Ligand in Wnt/β-Catenin Pathway Promoting Osteoblast-like Cell Differentiation

Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly in-creased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.     

Protective Effect of Hydroxysafflor Yellow A on Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Rats

Objective: To observe the effect of hydroxysafflor yellow A(HSYA), an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflammation and pulmonary fibrosis induced by bleomycin(BLM) in rats. Methods: Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone(DXM) group. Three doses of HSYA(35.6, 53.3, and 80.0 mg·kg~(–1)·day~(–1)) were intraperitoneally(i.p.) injected in rats for 3 weeks after BLM administration and DXM was used as the positive control(n=8 or 12). Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor(TNF)-α, interleukin(IL)-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin(α-SMA) protein distribution in rat lung tissue was observed by immunohistochemistry. Results: On the 7 th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure(PaO_2) increased(HSYA 80.0 mg·kg~(–1), P0.01) and CO_2 partial pressure(PaCO_2) decreased(HSYA 53.3, 80.0 mg·kg~(–1), P0.05). Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg·kg~(–1) groups than those in the model group(all P0.05). Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO_2(HSYA 53.3, 80.0 mg·kg~(–1), P0.01), and decreased PaCO_2(53.3 and 80.0 mg·kg~(–1), P0.05). Further, the mRNA expression of TGF-β1, α-SMA, and collagen Ⅰ as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes(53.3, 80.0 mg·kg~(–1), P0.05). Hematoxylin and eosin and Masson's trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups(53.3, 80.0 mg·kg~(–1), P0.05). Conclusion: HSYA could alleviate acute lung inflammation and chronic pulmonary fibrosis induced by BLM in rats.     

Effect and Mechanism of Ganoderma lucidum Polysaccharides on Human Fibroblasts and Skin Wound Healing in Mice

Objective: To investigate the effects of Ganoderma lucidum polysaccharides(GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. Methods: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide(MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type Ⅰ(CICP) and transforming growth factor-β1(TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. Results: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts(P0.05 or P0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group(P0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group(P0.05 or P0.01). Conclusion: A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.     

Role of DOR-β-arrestinl-Bcl2 Signal Transduction Pathway and Intervention Effects of Oxymatrine in Ulcerative Colitis

This study was aimed to investigate the role of the delta-opioid receptor （DOR）-β-arrestinl-Bcl-2 signal transduction pathway in the pathogenesis of ulcerative colitis （UC） and the intervention effects of oxymatrine on UC. Forty Sprague-Dawley rats were divided into nor- mal group, model group, oxymatrine-treated group and mesalazine-treated group （n=10 each） at ran- dom. The rat UC model was established by intra-colonic injection of trinitrobenzene sulfonic acid in the model group and two treatment groups. The rats in oxymatrine-treated group were subjected to intramuscular injection of oxymatrine [63 mg/（kg·day）] for 15 days, and those in mesalazine-treated group given mesalazine solution [0.5 g/（kg·day）] by gastric lavage for the same days. Animals in normal group and model group were administered 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the rats were sacrificed for the removal of colon tissues. The expres- sion levels of DOR, β-arrestinl and Bcl-2 were determined in colon tissues by immunohistochemistry and real-time quantitative polymerase chain reaction （RT-PCR）, respectively. It was found that the expression levels of DOR, [3-arrestinl and Bcl-2 protein and mRNA were significantly increased in the model group as compared with the other groups （P〈0.05）. They were conspicuously decreased in both mesalazine-treated and oxymatrine-treated groups in contrast to the model group （P〈0.05）. No statistically significant difference was noted in these indices between mesalazine- and oxyma- trine-treated groups （P〉0.05）. This study indicated that the DOR-β-arrestinl-Bcl-2 signal transduc- tion pathway may participate in the pathogenesis of UC. Moreover, oxymatrine can attenuate the de- velopment of UC by regulating the DOR-β-arrestin 1-Bcl-2 signal transduction pathway.     

Role of β2-adrenoceptor-β-arrestin2-nuclear factor-κB signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis

Objective:To investigate the β2-adrenoceptor(β2AR)-β-arrestin2-nuclear factor-κB(NF-κB) signal transduction pathway and the intervention effects of oxymatrine in a rat model of ulcerative colitis.Methods: Forty SD rats were randomly divided into four groups,which included the normal control group,the model group, the mesalazine group and the oxymatrine treatment group,with 10 rats per group.Experimental colitis induced with trinitrobenzene sulfonic acid(TNBS) was established in each group except the normal control group.The rats in the oxymatrine treatment group were treated with intramuscular injection of oxymatrine 63 mg/(kg·d) for 15 days and the rats in the mesalazine group were treated with mesalazine solution 0.5 g/(kg·d) by gastric lavage for 15 days. The rats in the normal control group and model group were treated with 3 mL water by gastric lavage for 15 days. Diarrhea and bloody stool were carefully observed.Histological changes in colonic tissue were examined on day 7 in 2 rats per group that were randomly selected.The expression of β2AR,β-arrestin2 and NF-κB p65 in colon tissue and spleen lymphocytes were detected with immunohistochemistry and Western immunoblotting techniques on day 16 after fasting for 24 h.Six rats died of lavage with 2 each in the normal control,the model group and the mesalazine group;and were not included in the analysis.Results:The rats in the model group suffered from looser stool and bloody purulent stool after modeling.But in the oxymatrine and mesalazine groups,looser stool and bloody purulent stool reduced after treatment.And the colonic wall in the model group was thickened and the colon length shortened.The colon mucosa was congested in multiple areas with edema,erosion,superficial or linear ulcer and scar formation,while the intestinal mucosa injury reduced in the mesalazine and oxymatrine groups(P<0.01).In colonic mucosa and in spleen lymphocytes,compared with the normal control group,the expression of NF-κBp65 were significantly increased(P<0.01) in the model group while the expressions ofβ2AR andβ-arrestin2 were significantly decreased(P<0.01).Compared with the model group,the expression of NF-κBp65 was significantly decreased in the mesalazine group(P<0.01) and oxymatrine treatment group(P<0.01) while the expressions of β2AR and β-arrestin2 were significantly increased(P<0.01).There were no statistically significant differences in the expression of β2AR,β-arrestin2 and NF-κBp65 between the mesalazine group and oxymatrine group(P>0.05).Conclusions:The β2AR-β-arrestin2-NF-κB signal transduction pathway participated in the pathologic course of ulcerative colitis.Oxymatrine attenuated ulcerative colitis through regulating the β2AR-β-arrestin2-NF-κB signal transduction pathway.     

Expression of β-catenin in renal cell carcinoma

Objective To investigate the expression of β-catenin and its mRNA in renal cell carcinoma.Methods Twenty-six cases with renal cell carcinoma (RCC) were studied by immunohistoche mistry, Western blot and RT-PCR.Results We found the expression of β-catenis is higher in cancer tissues than in norma l kidney tissues and the level of β-catenin is associated with the tumor stage . Its expression in tumor of pT3 and pT4 is obviously higher than pT1 and pT2 ( P&lt;0.01). That is to say, there was an overexpression of β-catenin protei n in RCC and its level was related to the tumor stage, but the expression of β -catenin mRNA had no difference between tumor tissue and normal tissue.Conclusion β-catenin may be related to the occurrence and progress of RCC.     

Effect of mexican tea herb and pilular adina herb on concrescence of gastric mucosa in experimental gastric ulcer rats

Objective:To study the effect and mechanism of mexican tea herb and pilular adina herb(abbreviated to MP) on concrescence of gastric mucosa in experimental gastric ulcer rats by observing the changes of epidermal growth factor(EGF),nitrogen monoxidum(NO) and expression of epidermal growth factor receptor(EGFR).Methods:The rat ulcer model was established by 100% glacial acetic injection into the subserosa.The ulcer index(UI) was measured by sliding caliper.The levels of NO and EGF in tissue and serum were measured by the nitrate reductase method and enzyme-linked immunosorbent assay,respectively.The expression of EGFR in the mucosa around the ulcer was detected by the immunohistochemical assay and microimage analysis system.Results:(1) Compared with the model group,UI of MP groups(10,15 and 20 mg·kg-1·d-1) and ranitidine group was lower(P<0.05 or P<0.01),the levels of NO and EGF in the tissue and serum were higher(P<0.05),the thickness of regenerated mucous membrane increased,and the width loss of lamina muscularis mucosa decreased(all P<0.05).(2) The expression of EGFR is weakly positive in gastric mucosa cells in the normal group,mainly in the cytoplasm and cytomembrane.In the model group,the expression of EGFR was mainly in epithelial cells in cervical part and basilar part of gastric gland around the ulcer margin,and the number of cells with EGFR weakly positive expression was more than that in the normal group.Compared with that in the normal and model groups,the number of cells with EGFR positive in MP groups and ranitidine group increased(all P<0.05),with weakly positive expression.Conclusion:MP can protect gastric mucosa,cure gastric ulcer,restrain the secretion of gastric acid,and boost multiplication,differentiation,migration and repair of the endothelial cell by promoting the secretion of NO and EGF,and increasing the expression of EGFR of gastric mucosa epithelial cells.     

Astragaloside Ⅳ inhibited hypoxia-enhanced proliferation and migration of human pulmonary arterial smooth muscle cells through modulating Wnt/β-catenin signaling pathway

Objective This study aims to determine the effects of astragaloside Ⅳ (AS-Ⅳ) treatment on the viability, proliferation and migration of hypoxia-stimulated human pulmonary arterial smooth muscle cells (PASMCs), and to explore the underlying molecular mechanisms. Methods The mRNA and protein expression levels of proliferating cell nuclear antigen (PCNA) were determined qRT-PCR and Western blot assays, respectively. Cell viability and cell proliferation were determined by CCK-8 and 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assays, respectively. The cell migration was determined by Transwell migration assay. Results Hypoxia stimulation up-regulated the mRNA and protein expression of PCNA in PASMCs (P<0.05); hypoxia stimulation significantly promoted PASMC viability and proliferation (P<0.05), and also increased the migration of PASMCs (P<0.05). AS - Ⅳ concentration-dependently down-regulated the mRNA expression and protein expression of PCNA (P<0.05), inhibited the viability, proliferation and migration of PASMCs under hypoxia (P<0.05). Hypoxia stimulation activated the Wnt/β-catenin signaling in PASMCs; while AS-Ⅳ concentration-dependently repressed the Wnt/βcatenin signaling in the hypoxia-stimulated PASMCs (P<0.05). Moreover, the treatment of XAV393, a Wnt/β - catenin inhibitor, attenuated the hypoxia-induced increase in the viability, proliferation and migration of PASMCs (P<0.05). The treatment of LiCl, a Wnt/β - catenin activator, restored the mRNA and protein expression levels of PCNA in the hypoxia-stimulated PASMCs with AS-Ⅳ treatment (P<0.05). The inhibitory effects of AS-Ⅳ treatment on the viability, proliferation and migration of PASMCs under hypoxia was attenuated by LiCl treatment (P<0.05). Conclusion Our results indicate that AS-Ⅳ reversed hypoxia-induced proliferation and migration of human pulmonary artery smooth muscle cells, which may be by regulating the Wnt/β-catenin signaling pathway. © 2022 China Tropical Medicine. All rights reserved.