Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway.

BACKGROUND: Constitutive activation of STAT3 is one of the major oncogenic pathways involved in the development of various types of malignancies including colorectal cancer (CRC); and thus becomes a promising therapeutic target. Spica Prunellae has long been used as an important component in many traditional Chinese medicine formulas to clinically treat CRC. Previously, we found that Spica Prunellae inhibits CRC cell growth through mitochondrion-mediated apoptosis. Furthermore, we demonstrated its anti-angiogenic activities in vivo and in vitro. To further elucidate the precise mechanism of the potential tumoricidal activity of Spica Prunellae, using a CRC mouse xenograft model, in this study we evaluated its therapeutic efficacy against CRC and investigated the underlying molecular mechanisms. METHODS: CRC mouse xenograft model was generated by subcutaneous injection of human colon carcinoma HT-29 cells into nude mice. Animals were given intra-gastric administration with 6 g/kg of the ethanol extract of Spica Prunellae (EESP) daily, 5 days a week for 16 days. Body weight and tumor growth were measured every two days. Tumor growth in vivo was determined by measuring the tumor volume and weight. HT-29 cell viability was examined by MTT assay. Cell apoptosis and proliferation in tumors from CRC xenograft mice was evaluated via immunohistochemical staining (IHS) for TUNEL and PCNA, and the intratumoral microvessel density (MVD) was examined by using IHS for the endothelial cell-specific marker CD31. The activation of STAT3 was evaluated by determining its phosphorylation level using IHS. The mRNA and protein expression of Bcl-2, Bax, Cyclin D1, VEGF-A and VEGFR2 was measured by RT-PCR and IHS, respectively. RESULTS: EESP treatment reduced tumor volume and tumor weight but had no effect on body weight change in CRC mice; decreased HT-29 cell viability in a dose-dependent manner, suggesting that EESP displays therapeutic efficacy against colon cancer growth in vivo and in vitro, without apparent toxicity. In addition, EESP significantly inhibited the phosphorylation of STAT3 in tumor tissues, indicating its suppressive action on the activation of STAT3 signaling. Consequently, the inhibitory effect of EESP on STAT3 activation resulted in an increase in the pro-apoptotic Bax/Bcl-2 ratio, decrease in the expression of the pro-proliferative Cyclin D1 and CDK4, as well as down-regulation of pro-angiogenic VEGF-A and VEGFR-2 expression. Finally, these molecular effects led to the induction of apoptosis, the inhibition of cell proliferation and tumor angiogenesis. CONCLUSIONS: Spica Prunellae possesses a broad range of anti-cancer activities due to its ability to affect STAT3 pathway, suggesting that Spica Prunellae could be a novel potent therapeutic agent for the treatment of CRC.

[Study on function of VEGF pathway in promoting angiogenesis with Xuefu Zhuyu Tang].

OBJECTIVE: To investigate the function of VEGF pathway in promoting angiogenesis with Xuefu Zhuyu Tang (XFZYT). METHOD: Serum pharmacology technique was adopted to treat endothelial cells ECV304 with XFZYD containing serum and blank serum with concentrations of 1.25%, 2.5% and 5%. Methyl thiazolyl tetrazolium (MTT) assay, boyden chamber migration assay and in vitro vessel formation assay were used to test endothelial cell proliferation, migration and angiogenesis capacities. ELISA, immunohistochemistry and RT-PCR were used to detect secretion and expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR2). RESULT: XFZYT-CS with a concentration of 1.25% only affected angiogenesis capacity in vitro. XFZYT-CS with a concentration of 2. 5% promoted cell proliferation, migration and angiogenesis capacities and significantly increased VEGF level and VEGF/VEGFR2 expressions. XFZYT-CS with a concentration of 5% only up-regulated intracellular VEGF expression and thereby improving endothelial cell proliferation, migration and angiogenesis. CONCLUSION: XFZYT can induce endothelial cell proliferation, migration and angiogenesis by up-regulating VEGF-VEGFR2 pathway, which partially proves its angiogenesis effects.

Xinshuitong Capsule extract attenuates doxorubicin-induced myocardial edema via regulation of cardiac aquaporins in the chronic heart failure rats.

Doxorubicin (Dox), an effective antineoplastic drug, was limited use for cardiotoxicity. Xinshuitong Capsule (XST), a patented herbal formula, showed desirable beneficial effects in the treatment of chronic heart failure (CHF) patients. However, the drug on Dox-induced cardiotoxicity remains unclear. Ninety male Sprague-Dawley rats were randomized into two groups: 15 rats were selected as the normal group and 75 rats were injected intraperitoneally with Dox to establish CHF rat models, the success ones were randomly divided into five groups: low XST (LXST), medium XST (MXST) or high XST (HXST) (4.9, 9.8, or 19.6 g/kg d) administrated intragastrically twice a day for 4 weeks, with the captopril-treated group and the model group as comparison. The model group showed the cardiac functions generally impaired, and CHF mortality rate higher (47%) than those in the XST-treated groups (averaged 24%, P < 0.05). Compared with XST-treated groups, myocardial remodeling, inflammation and desarcomerization, and higher water content more severe in the cardiac tissue in the model group (P < 0.05), which was associated with higher expressions of mRNA or protein levels of AQP1, 4 and 7. Dox-impaired cardiac functions, cardiac remodeling and myocardial edema could be dose-dependently reverted by XST treatment. XST could inhibit AQP1, 4 and 7 at mRNA levels or at protein levels, which was associated with the attenuation of myocardial edema and cardiac remodeling, decreasing the ventricular stiffness and improving the cardiac functions and rats' survival. AQPs is involved in cardiac edema composed one of the mechanisms of Dox-induced cardiotoxicity, XSTvia inhibition of AQPs relieved the Dox-induced side effects.

Establishment of a stable hepatic metastasis mouse model of murine colorectal cancer by microsurgical orthotopic implantation.

BACKGROUND: Liver metastasis is a common cause of death from colorectal cancer (CRC). In this paper we developed a liver metastasis mouse model by microsurgical orthotopic implantation (MSOI) to illuminate the CRC progression with an eye toward developing effective drug treatment. METHODS: Murine colon carcinoma CT-26 cells were cultured and then injected to male BALB/c athymic nude mice right flank to generate subcutaneous implantation tumor with 2×107 CT-26 cell suspension in DMEM. Tumor tissue at an average size of 1 cm3 was injected into another nude mice right flank with 20-gauge inoculating needle. Between fourth and sixth generations, tumor tissue sewn into the cecal surface establishes orthotopic transplanted CRC model by MSOI. Then on the 7th, 14th, 21st and 35th day, body weight, abdomen circumference, volume of ascites and local tumor weight were observed and weighed. On the 21st day and 35th day, local tumor rate was calculated, and metastatic tumors of other organs were observed. Tumor tissue was stained by HE for pathologic analysis. RESULTS: On the 35th day, body weight and abdomen circumference of the model group were significantly higher than the control group (P<0.01). Local tumor weight increased rapidly from the 21st d to the 35th d (P<0.01), and take rate was high (100%). Metastatic tumor appeared only in liver on the 21st day and then invaded to liver, stomach, retroperitoneal lymph node and abdominal wall on the 35th day. The metastatic rate of liver tumor respectively was 83.3% and 100% on the 21st day and 35th day, but liver function remained normal. Pathologic analysis showed that colorectal tumor invaded the normal tissue of liver, abdominal wall and stomach. CONCLUSIONS: A stable hepatic metastasis mouse model of murine CRC was established by MSOI.

Tougu Xiaotong capsule exerts a therapeutic effect on knee osteoarthritis by regulating subchondral bone remodeling.

Previous studies have shown that Tougu Xiaotong capsule (TGXTC) has therapeutic effects on knee osteoarthritis (OA) through multiple targets. However, the mechanisms of action underlying its regulation of subchondral bone reconstruction remain unclear. In this study, we investigated the effects of TGXTC on subchondral bone remodeling. Eighteen six-month-old New Zealand white rabbits of average sex were randomly divided into the normal, model and TGXTC groups. The rabbit knee OA model was induced by a modified Hulth's method in the model and TGXTC groups, but not the normal group. Five weeks postoperatively, intragastric administration of TGXTC was performed for four weeks. After drug administration, the medial femoral condyle and tibia were prepared for observation of cartilage histology via optical microscopy and micro-computed tomography, the serum was collected for biochemical parameters assay and the subchondral bone isolated from the lateral femoral condyle was collected for detection of IL-1ß and TNF-α mRNA and protein by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results showed that treatment with TGXTC significantly mitigated cartilage injury and subchondral bone damage, improved the parameter of subchondral trabecular bone, decreased alkaline phosphatase and tartrate-resistant acid phosphatase activity, and significantly reducing the osteoprotegerin/receptor activator of nuclear factor-κB ligand ratio, reduced the expression of IL-1ß and TNF-α mRNA and protein. These results suggest that TGXTC could delay the pathological development of OA by regulating subchondral bone remodeling through regulation of bone formation and bone resorption and its relating inflammatory factors, and this may partly explain its clinical efficacy in the treatment of knee OA.

Inflammatory cytokines via up-regulation of aquaporins deteriorated the pathogenesis of early osteoarthritis.

BACKGROUND: Inflammatory cytokines enhanced the progress of the pathogenesis of osteoarthritis, however the mechanisms remain unclear. The objective is to determine aquaporins (AQPs) in the pathogenesis of osteoarthritis. METHODS AND FINDINGS: Primary rat articular chondrocytes were treated with IL-1ß to mimic the early stage of osteoarthritis in vitro. Early osteoarthritis animal model was established by intra-articular injection of 4% papain. Micro- or ultra-structure histopathologic changes, cell viability, apoptosis cells and cell membrane permeability, locations and expressions of AQP1 and AQP3 and matrix were detected in the cartilage or in the chondrocytes of knee. IL-1ß could reduce the chondrocytes viability, increase the apoptosis cells, and also impair the cell membrane and organelles. IL-1ß significantly induced the up-regulation of AQP1 and AQP3 in the chondrocytes. In the chondrocytes, AQPs were mainly clustered in both membrane and perinuclear region of cytoplasm, while higher AQPs were detected in the superficial and middle layers of the cartilage. With the up-regulation of AQPs, the cartilage matrix was considerably decreased in both the chondrocytes and in the osteoarthritis cartilage. In the early osteoarthritis rat model, serum and synovial fluid confirmed that higher IL-1ß could increase the expressions of AQPs, and decrease the cartilage matrix in both the chondrocytes and the cartilage. CONCLUSIONS: Inflammatory cytokine IL-1ß via up-regulation of AQPs caused the abnormal metabolism of water transport and loss of the cartilage matrix in the chondrocytes, and ultimately exacerbated the pathogenesis of early osteoarthritis. Therefore, AQPs may be a candidate therapeutic target for prevention and treatment of osteoarthritis.

[Experimental study on effect of Liuwei Dihuang Pill on HSC from mouse marrow].

OBJECTIVE: To investigate the effect of Liuwei Dihuang Pill on the number, the surface marker, cell cycle and colony formation of HSC from mouse marrow. METHODS: Old Kunming mice were randomly divided into 4 groups: the control group, low, middle and high dose of Liuwei Dihuang Pill group. Then we separated the HSC from marrow after 7 days fed with saline or Liywei Dihuang Pill respectively, numerated monocyte, detected the surface marker and cell cycle of the HSC by FACS and tested the colony forming by semisolid media culture. RESULTS: Among the four groups, there was no obvious difference in the number of MNC, suspended cell and colony. The expression of Sca-1 and CD34 increased in the low and middle dosage group, it meant that the number of HSC elevated by low and middle dosage medicine. The ability of cell proliferation was also higher in the three dosage groups. CONCLUSION: Liuwei Dihuang Pill activates HSC by increasing the number, proliferation and function of more primitive HSC.

Ultrastructural change of the subchondral bone increases the severity of cartilage damage in osteoporotic osteoarthritis of the knee in rabbits.

Osteoporotic osteoarthritis is a phenotype of osteoarthritis (OA) manifested as fragile and osteoporotic subchondral bone. However, the ultrastructural features of subchondral bone in osteoporosis OA have not been determined. The study was aimed to investigate the ultrastructural dynamic changes of subchondral bone in osteoporotic OA model and how the ultrastructural damage in the subchondral bone caused by osteoporosis deteriorated the cartilage damage in OA. Eighteen rabbits were equally randomized to three groups, including the control, the OA and the osteoporotic OA groups. The structural changes of cartilage were evaluated by HE and safranin-O fast green staining, the Mankin's grading system was used to assess the stage of OA progression. And microstructural or ultrastructural changes in subchondral bone were assessed by micro-computed tomography or by scanning electron microscopy. According to the changes of cartilage histopathology, the OA group was in the early pathological stage of OA while the osteoporotic OA group was in the middle stage of OA based on Mankin's grading system. In addition, the damage of cartilage surface, reduction in the number of chondrocytes and the matrix staining were more increased in the osteoporotic OA group compared to the OA group. Compared to the OA group, the subchondral bone in the microstructure and ultrastructure in the osteoporotic OA group showed more microfracture changes in trabecular bone with more destructions of the tree-like mesh. Moreover, the collagen fibers were random rough with a fewer amount of bone lacunae in subchondral cortical plate in the osteoporotic OA group compared to the OA group. These findings indicated that the subchondral bone ultrastructure in the osteoporotic OA model was characterized by the destruction of the network structure and collagen fibers. The subchondral bone ultrastructural damage caused by osteoporosis may change mechanical properties of the upper cartilage and aggravate OA cartilage. Therefore, early diagnosis and treatment of osteoporosis is of great significance to prevent early OA from further developing osteoporotic OA.

[Experimental study on effect of Xuefu Zhuyu Decoction on bone marrow hematopoietic stem cells of mice].

OBJECTIVE: To investigate the effect of Xuefu Zhuyu Decoction (XFZYD) on the number, phenotype, cell cycle and colony formation of bone marrow hematopoietic stem cells (HSC) in mice. METHODS: Kunming mice were randomly divided into 4 groups: the control group, the low- (3.25 g/kg), middle- (6.5 g/kg) and high-dose (13.0 g/kg) XFZYD groups. After they were medicated by gastrogavage respectively with saline or corresponding dose of XFZYD for 7 days, their bone marrow HSC were separated and counted. The phenotype Sca and cell cycle of HSC were detected by flow cytometer, and the colony formation was determined with semisolid methyl media culture. RESULTS: No obvious difference in the number of mononuclear cell, suspended cell and colony production was found among all the groups (P > 0.05); while the expression of CD34 and Sca-1 increased in the low-dose XFZYD group, but in the middle-dose XFZYD group increase only showed in Sca-1 expression. CONCLUSION: XFZYD plays a role of removing blood stasis and promoting regeneration through improving hematopoietic function by means of increasing the number and enhancing the function of premature HSC.

Pien Tze Huang Gan Bao ameliorates carbon tetrachloride-induced hepatic injury, oxidative stress and inflammation in rats.

Liver damage results from a variety of insults, including hepatitis and chemical toxicity from alcohol, drugs and other toxins. The present study evaluated the hepatoprotective effects and potential mechanisms of action of the Traditional Chinese Medicine Pien Tze Huang Gan Bao (GB) in a rat model of carbon tetrachloride (CCl4)-induced liver injury. Sixty male Sprague-Dawley rats were randomly divided into six different groups: i) Control, ii) CCl4 injury model and groups treated with iii) silymarin as a positive drug control, iv) 150 mg/kg GB, v) 300 mg/kg GB and vi) 600 mg/kg GB. Control rats received no treatment, while the remaining ones were intraperitoneally injected with CCl4 (2 ml/kg) to induce acute liver disease. Silymarin or GB was orally administered prior to CCl4 treatment in various treatment groups for 7 days. Animals were sacrificed 24 h post-CCl4 injection. It was revealed that GB significantly reduced serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase and total bilirubin levels in the serum induced by CCl4. BG also prevented CCl4-induced changes in liver tissues, as revealed by histopathological analysis. CCl4-induced reductions in endogenous liver antioxidant enzyme activities of superoxide dismutase, glutathione and glutathione peroxidase as well as increases in malondialdehyde and thiobarbituric acid reactive substances were inhibited by GB treatment. Activated NF-κB in liver tissues was also significantly increased by CCl4, which was attenuated by GB as indicated by immunohistochemical and PCR analysis. Furthermore, CCl4-mediated increases in the inflammatory factors tumor necrosis factor-alpha and interleukin-1ß secretion into the serum and their expression in liver tissues were reversed following GB treatment, as revealed by ELISA and PCR, respectively. These findings suggested that GB protects against CCl4-induced hepatic injury, inflammation and oxidative damage in rats and may be useful in future clinical application of liver injury and disease.

Anti-angiogenic effect of Livistona chinensis seed extract in vitro and in vivo.

The present study aimed to detect the impact of the ethanol extract of the Livistona chinensis seed (EELC) on angiogenesis in human umbilical vein endothelial cells (HUVECs). A chorioallantoic membrane (CAM) assay was used to detect the anti-angiogenic activity of EELC in vivo. In vitro, the effect of EELC on the proliferation, migration and angiogenesis of HUVECs was determined by an MTT assay, a wound healing assay and a tube formation assay, respectively. The vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR)-2 protein and mRNA level were measured with ELISA and reverse transcription-semi-quantitative polymerase chain reaction. It was observed that EELC significantly decreased the formation of new vessels in the CAM assay. EELC inhibited the proliferation and migration of HUVECs. The extent of tube formation by HUVECs was also reduced by EELC. In addition, EELC treatment reduced the level of VEGF-A and VEGFR-2 mRNA and protein. The results suggest that EELC inhibits tumor angiogenesis through inhibiting the proliferation and migration of HUVECs, and by downregulating VEGF and VEGFR.

[Angiogenesis promoting effects of Chinese herbal medicine for activating blood circulation to remove stasis on chick embryo chorio-allantoic membrane].

OBJECTIVE: To observe the angiogenesis promoting effects of clinical common used Chinese herbal medicines (CHM) for activating blood circulation to remove stasis on chick embryo chorio-allantoic membrane (CAM). METHODS: Chicken CAM model was established and mice blood serum containing different kinds of medicines, including Radix Peaoniae rubra, Radix Angelicae sinensis, Flos Carthami, Rhizoma Chuanxiong, Radix Salviae miltiorrhizae, Astragalus membranaceus, and their complex prescriptions, Danggui Buxue Decoction, Xuefu Zhuyu Decoction, Xiongshao Capsule, was applied on it respectively to observe the condition of angiogenesis 72 hrs after incubation. Besides, the normal saline group, blank serum group, blank group and basic fibroblast growth factor (bFGF) group were set up for control. RESULTS: All the CHM applied and bFGF had the CAM angiogenetic promoting effect, among them, Radix Salviae Miltiorrhizae and the three complex prescriptions showed better effects than the three negative control groups in capillary formation and count, with the efficacy similar to that of bFGF. The effect of complex prescriptions was superior to that of single herb except Radix Salviae miltiorrhizae. CONCLUSION: Radix Salviae miltiorrhizae, Danggui Buxue Decoction, Xuefu Zhuyu Decoction and Xiongshao Capsule have good angiogenesis promoting effect on CAM. This study elucidated, from a certain aspect, the mechanism of action of CHM on ischemic diseases, and unfolded the scientific evidence of applying complex prescription.

Jiedu Xiaozheng Yin decoction inhibits hepatoma cell proliferation by inducing apoptosis via the mitochondrial-mediated pathway.

Jiedu Xiaozheng Yin decoction (JXY) is a type of Chinese traditional medicine, which has been used to treat various types of cancer. The present study explored the mechanisms underlying the anticancer activity of JXY. The effects of ethyl acetate extraction of JXY (EE-JXY) were evaluated on the HepG2 human hepatoma cell line in vitro and in vivo. Following treatment of the HepG2 cells with EE-JXY for 24 h, cell viability, apoptosis, mitochondrial membrane potential, caspase enzyme activity and the expression levels of apoptotic-associated proteins (Bcl-2 and Bax) were detected by MTT, flow cytometry, ELISA and western blotting respectively. In addition, HepG2 cells were subcutaneously transplanted into BALB/c nude mice, and the tumor bearing mice were treated with either EE-JXY (0.06 g/kg) or normal saline for 21 days. Tumor volume and weight were measured and recorded. The apoptotic index, and the expression levels of Bax and cytochrome c were determined with immunohistochemical staining. Treatment with EE-JXY inhibited the proliferation of HepG2 cells, and reduced cell viability in a dose-and time-dependent manner. Furthermore, EE-JXY induced HepG2 cell apoptosis, as demonstrated by a loss of plasma membrane asymmetry and externalization of phosphatidylserine, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Furthermore, EE-JXY inhibited tumor growth and increased the apoptotic index of tumors in tumor-bearing mice. In conclusion, the results of the present study suggest that JXY inhibits HepG2 cell proliferation through mitochondrion-mediated apoptosis, which may partially explain its anticancer activity.

Total alkaloids of Rubus alceifolius Poir inhibit tumor angiogenesis through suppression of the Notch signaling pathway in a mouse model of hepatocellular carcinoma.

Angiogenesis, which has a critical role in human tumor growth and development, is tightly regulated by the Notch signaling pathway. Total alkaloids are active components of the plant Rubus alceifolius Poir, which is used for the treatment of various types of cancer. A previous study by our group showed that the total alkaloids of Rubus alceifolius Poir (TARAP) induced hepatocellular carcinoma (HCC) cell apoptosis through the activation of the mitochondria-dependent pathway in vitro and in vivo, as well as inhibited angiogenesis in a chick embryo chorioallantoic membrane model. In the present study, to further analyze the specific mechanisms underlying the antitumor activity of TARAP, a HCC xenograft mouse model was used to assess the effect of TARAP on angiogenesis in vivo. TARAP was found to suppress the expression of vascular endothelial growth factor (VEGF) A and VEGF receptor-2 in tumor tissues, which resulted in the inhibition of tumor angiogenesis. In addition, TARAP treatment was observed to inhibit the expression of Notch1, delta-like ligand 4 and jagged 1, which are key mediators of the Notch signaling pathway. The present study identified that the inhibition of tumor angiogenesis through the suppression of the Notch signaling pathway may be one of the mechanisms through which TARAP may be effective in the treatment of cancer.

Pien Tze Huang inhibits liver metastasis by targeting TGF-ß signaling in an orthotopic model of colorectal cancer.

Metastasis is the leading cause of cancer-related mortality in almost all types of cancers, including colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a critical process during the metastatic cascade. This process may be a potential target for the diagnosis and treatment of CRC. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in treating various types of human malignancies, including CRC. Our published data suggest that PZH can induce apoptosis, as well as inhibit cell proliferation and tumor angiogenesis, thus suppressing CRC growth in vitro and in vivo. We evaluated the therapeutic efficacy of PZH against CRC metastasis using a CRC liver metastasis mouse model to further explore the mechanisms underlying the antitumor action of PZH. MTT, migration, and Matrigel invasion assays were used to assess the effect of PZH on cell viability, migration and invasion. We then established an orthotopic liver metastasis model of colon cancer using microsurgical techniques. Mice were intragastrically administered 234 mg/kg/day dose of either PZH or saline for 14 days. The body and tumor weights of the mice were measured after they were sacrificed. Moreover, we examined the effect of PZH inhibition on liver metastasis. Finally, EMT-related proteins and the TGF-ß signaling pathway were assessed using immunohistochemical staining (IHS). The present data revealed that PZH significantly inhibited the migration and invasion of CT-26 cells in a dose-dependent manner, which affirmed the inhibitory effect of PZH on CRC cell metastasis. No significant change was observed between the in vivo primary tumor growth and body weight. However, the control group had five cases of liver metastasis (5/6), whereas one case was found in the PZH group (1/6). Thus, PZH exhibited therapeutic efficacy against CRC metastasis without apparent toxicity. The inhibitory effect of PZH on EMT resulted in an increase in E-cadherin expression, as well as a decrease in N-cadherin expression. In addition, PZH significantly inhibited TGF-ß, as well as the phosphorylation of Smad2/3 and Smad4 in the tumor tissues, indicating its suppressive action on TGF-ß signaling. These molecular effects ultimately resulted in the inhibition of cancer cell EMT and tumor metastasis.

Protective effects of Tougu Xiaotong capsule on tumor necrosis factor-α-injured UMR-106 cells.

Tumor necrosis factor-α (TNF-α) plays an important role in the abnormal metabolism of osteoblasts (OBs), which leads to subchondral bone (SB) alterations in osteoarthritis. In the present study, Tougu Xiaotong capsule (TXC), a traditional Chinese medicine, was used to treat TNF-α-injured OB-like cells. The cellular viability, mortality and ultramicroscopic morphology were evaluated. Thereafter, the activity of alkaline phosphatase (ALP), secretion of osteocalcin (OCN) and mineralization of nodules were analyzed. The results showed that TXC treatment significantly promoted cell proliferation, reduced cellular mortality and improved cellular ultrastructure, particularly that of the endoplasmic reticulum and nucleus. These data indicate that TXC is able to promote cell growth, as well as prevent inflammation in OB-like cells. Furthermore, the activity of ALP, secretion of OCN and mineralization of nodules were accelerated, and the calcium content of the TNF-α-injured OB-like cells was promoted by TXC treatment. These results indicate that TXC protected the OB-like cells from TNF-α-induced injuries. This may be a potential mechanism through which TXC regulates SB remodeling in the clinical treatment of osteoarthritis.

[A study on cytokine levels and nitric oxide content in rabbit aqueous humor after lens extraction and intraocular lens implantation].

OBJECTIVE: To investigate the relationship among inflammatory reaction and cytokine levels, nitric oxide (NO) content in aqueous humor after intraocular lens implantation. METHODS: Eighteen New Zealand rabbits were divided randomly into 3 groups (each 6 rabbits): (1) control group, (2) extracapsular cataract extraction group (ECCE) and (3) ECCE and posterior chamber intraocular lens implantation group (ECCE + IOL). The inflammation of all experimental rabbit eyes were observed by a zoom-photo slit-lamp microscope 0, 1, 3, 7, 14, 30 days postoperatively, including corneal edema and anterior chamber exudation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) count and classification, as well as for NO(2)(-)/NO(3)(-) and cytokine assays, including interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha). Statistics were taken by SPSS software. RESULTS: (1) The anterior chamber exudation was the most serious and monocyte/macrophage in aqueous humor were the highest in ECCE + IOL group in postoperative 7 - 14 days. (2) The levels of IL-2, TNF-alpha and the content of NO(2)(-)/NO(3)(-) in aqueous humor of ECCE + IOL group were higher than that in ECCE group and control group in the postoperative 1 - 14 days respectively, and they increased to their peak values at the postoperative 3 - 7 days and decreased gradually after postoperative two weeks. (3) The change regularity of IL-2, TNF-alpha, NO(2)(-)/NO(3)(-) and inflammatory reaction in each group were basically similar, i.e. the more serious the reaction, the higher the levels of the contents. CONCLUSION: The intraocular inflammation after intraocular lens implantation is closely related to the changes of cytokine levels and NO content in aqueous humor.

[Effect of Bugu Mixture on all-trans retinoic acid-induced apoptosis of bone marrow stromal cells].

OBJECTIVE: We used the SD rat's bone marrow stromal cells (BMSCs) cultured in vitro to observe the effects of Bugu Mixture on the apoptosis and to explore the molecular biologic mechanism of the treatment of osteoporosis with Bugu Mixture. METHODS: BMSCs were separated from the bones of the extremities of SD rats in vitro. The morphologic changes, the apoptosis cell cycles, the mitochondrion membrane potential changes, and the Bcl-2 and Bax gene expression were observed, and the effects of Bugu Mixture on the course of cell apoptosis were evaluated. RESULTS: The earlier use of Bugu Mixture could decrease the cells blocked in G0/G1 phase, and promote their synthesis of DNA in S phase. The expression of Bcl-2 was higher in the Bugu Mixture group than that in the all-trans retinoic acid (ATRA) induced group, and the expression of Bax was lower in the Bugu Mixture group than that in the ATRA induced group. The mitochondrion membrane potential descended significantly in the Bugu Mixture group than that in the ATRA induced group. CONCLUSION: The mechanism of the treatment of osteoporosis with Bugu Mixture is that the earlier use of Bugu Mixture can decrease the amount of apoptostic cells induced by ATRA, thus promoting the cell mitosis and restraining the apoptosis. It can also act as a protector to Bcl-2 located on the mitochondrion membrane. By preventing the transferring of the Bax protein from cell-plasma to mitochondrion membrane, it takes the advantage of Bcl-2 in forming Bcl-2/Bax homodimer so as to prevent the opening of the permeability transition pore to avoid the changing of mitochondrion membrane potential and the destruction of biosynthesis caused by the mitochondrion release of apoptosis inducing factors and to reach the objective of restraining apoptosis.

Livistona chinensis seeds inhibit hepatocellular carcinoma angiogenesis in vivo via suppression of the Notch pathway.

Livistona chinensis seeds have been used for centuries to clinically treat various types of cancer. Our published data suggest that Livistona chinensis seeds are able to inhibit hepatocellular carcinoma (HCC) growth in vitro and in vivo via promotion of mitochondrial-dependent apoptosis. To further elucidate the molecular mechanisms of its antitumor activity, in the present study, we used an HCC xenograft mouse model to evaluate the effect of an ethanol extract of Livistona chinensis seeds (EELC) on tumor angiogenesis and on the activation of the Notch pathway. Intratumoral microvessel density (MVD) in HCC xenograft mouse tumors was evaluated via immunohistochemical (IHC) staining for CD31. The mRNA and protein expression of vascular endothelial growth factor A (VEGF-A), VEGFR-2, Notch, Dll4 and Jagged1 was evaluated using RT-PCR and IHC, respectively. We found that EELC profoundly reduced MVD in the HCC mouse tumors, demonstrating the in vivo inhibitory effect of EELC on tumor angiogenesis. In addition, EELC treatment reduced the expression of VEGF-A and VEGFR-2 in tumor tissues. Furthermore, EELC treatment inhibited the expression of Notch, Dll4 and Jagged1. Our findings suggest that Livistona chinensis seeds inhibit tumor angiogenesis through suppression of the Notch pathway.

Livistona chinensis seed suppresses hepatocellular carcinoma growth through promotion of mitochondrial-dependent apoptosis.

The Livistona chinensis seed has been used for centuries to clinically treat various types of cancer. However, the precise mechanism of its anticancer activity remains to be elucidated. In the present study, we evaluated the efficacy of the ethanol extract of Livistona chinensis seed (EELC) against tumor growth using a hepatocellular carcinoma (HCC) mouse xenograft model and an HCC cell line, HepG2, and investigated the molecular mechanisms mediating its biological activities. We found that EELC inhibited HCC growth both in vivo and in vitro, without apparent signs of toxicity. In addition, EELC treatment resulted in the induction of HCC cell apoptosis. Moreover, EELC-induced apoptosis was accompanied by the loss of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase in the pro-apoptotic Bax/Bcl-2 ratio. Our findings suggest that promotion of mitochondrial-dependent apoptosis in cancer cells may be one of the mechanisms by which the Livistona chinensis seed is effective in cancer treatment.