ABSTRACT
Objective:To observe the effect of Danggui Buxuetang on lung histopathology and protein kinase D1 (PKD1), nuclear transcription factor-κB (NF-κB) and manganese superoxide dismutase (MnSOD)-mediated oxidative stress pathway in rats with pulmonary fibrosis induced by bleomycin, so as to explore the mechanism of intervention of pulmonary fibrosis.Method:Thirty-two male SPF SD rats were randomly divided into sham operation group, model group, Danggui Buxuetang group and prednisone group, with 8 rats in each group. Except the sham operation group, the other groups were prepared through the intratracheal instillation with bleomycin. After modeling for 24 h, the rats of Danggui Buxuetang group were administered with Danggui Buxuetang (0.81 g·kg-1). The rats of prednisone group were given aqueous solution of prednisone (0.005 g·kg-1). The rats of sham operation group and model group were given the same volume of saline. After 14 days of administration, blood was collected from the femoral artery, serum was separated, and the lungs were taken by thoracotomy. The pathological changes of rat lung tissues were observed by hematoxylin-eosin staining (HE) and Masson trichrome staining, and graded by Szapiel score and Ashcroft score at the same time. The content of serum malondialdehyde (MDA), and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) were determined. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to measure mRNA and protein expressions of PKD1, NF-κB, MnSOD.Result:Compared with the rats in sham operation group, the rats in model group had higher Szapiel scores and Ashcroft scores (P<0.05), higher serum MDA content , but lower SOD, CAT and GSH-Px activities(P<0.01), moreover, the rat lung tissues in model group had higher mRNA and protein expressions of PKD1, NF-κB and MnSOD (P<0.01) than those in sham operation group. Compared with the rats in model group, the Szapiel scores and Ashcroft scores of the rats in Danggui Buxuetang group were decreased significantly（P<0.05）. The serum MDA content was decreased significantly, and SOD, CAT, GSH-Px activities were increased, whereas mRNA and protein expressions of PKD1, NF-κB, MnSOD in the rat lung tissues were decreased（P<0.05，P<0.01）.Conclusion:Danggui Buxuetang can reduce the degree of pulmonary fibrosis by regulating the anti-oxidation pathway of PKD1/NF-κB/MnSOD mitochondrial nucleus and improving the body's antioxidant capacity.
ABSTRACT
Aim To investigate the effect of protein kinase Dl (PKD1) on myocardial inflammation and apoptosis after myocardial infarction and to analyze its molecular mechanism. Methods Forty-five male Wistar rats were randomly divided into three groups: sham-operated group, model group and PKD1 group. Rat models of myocardial infarction were reproduced by classical left coronary artery ligation in model group and PKD1 group, while rats in sham group were operated without ligation of coronary artery. The parameters of hemodynamics in rats were assessed, the morphological changes of myocardial tissue were analyzed by HE staining, the apoptotic changes of myocardial cells were analyzed by TUNEL staining, immunohistochemical staining and Western blot, while the changes of myocardial inflammation were analyzed by immunoblotting and real-time quantitative PCR. Results Compared with model group, PKD1 could improve the hemodynamic indexes in rats with myocardial infarction, reduce the injury caused by myocardial infarction, inhibit apoptosis in myocardial tissue, up-regulate the protein expression of Bcl-2, down-regulate the protein expression of caspase-3, Bax, TLR4, TN-C, NF-ΚB p50, NF-ΚB p65 and decrease the mRNA expression of IL-1, NF-ΚB p50 and NF-ΚB p65, and all the differences were statistically significant (P <0. 01). Conclusion PKD1 might have the biological function of inhibiory effect on inflammation and apoptosis induced by myocardial infarction injury.
ABSTRACT
OBJECTIVE@#To observe the effect of protein kinase D1 (PKD1) on the growth and metabolism of oral squamous cell carcinoma HSC-4 cells and related molecular mechanisms in the tumor microenvironment.@*METHODS@#HSC-4 cell lines were transfected with shRNA plasmids. Three groups (Wild, control-shRNA, and PKD1-shRNA) were cultured under acidic or hypoxic environment for a certain time. Western blot was used to detect the expression of autophagy-related and glycolytic-related proteins. The proliferation changes were detected by CCK-8 kits.@*RESULTS@#The PKD1-knockdown HSC-4 cell line was established. PKD1 silencing increased autophagy activity. Under hypoxic and acidic conditions, the PKD1-knockdown HSC-4 cells showed lower proliferation than the parental cells. PKD1-knockdown also decreased the expression of hypoxia induciblefactor 1α (HIF-1α) and pyruvate kinase M2 (PKM2).@*CONCLUSIONS@#Under hypoxic and acidic conditions, PKD1 gene silencing can increase apoptotic autophagy activity. Downregulated PKD1 gene expression can reduce the glycolysis of oral squamous cell carcinoma cells and inhibit tumor cell proliferation. This study revealed the important role of PKD1 in the metabolism and growth of oral squamous cell carcinoma, making it a possible target for the treatment of oral squamous cell carcinoma.
Subject(s)
Humans , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Proliferation , Hypoxia-Inducible Factor 1, alpha Subunit , Mouth Neoplasms , Protein Kinases , Tumor MicroenvironmentABSTRACT
OBJECTIVE This study aimed to investigate the role and mechanism of Astragaloside IV (AS-IV)in rats with myocardial infarction.METHODS The myocardial infarction model was established by ligation of the left anterior descending artery. The rats were randomly divided into sham, DMSO, model group, AS-IV and CID755673 groups. The rats were sacrificed 4 weeks later, and segmental heart samples were used for hematoxylin and eosin staining and masson staining. The expression of PKD1, HDAC5 and VEGF were analyzed using immunohistochemistry, reverse transcription poly-merase chain reaction and western blot. RESULTS Compared with the sham operation and DMSO groups,morphology of myocardium in model group was disordered,accompanied with necrotic myocar-dial cells and obvious collagen tissues. After treatment with AS-IV, the morphology of myocardium was obviously improved, and the number of new blood vessels increased significantly. However, after treatment with CID755673, the myocardial tissue of rats became disordered again, the necrotic cells increased, and some vessels closed. The expression levels of PKD1, HDAC5 and VEGF mRNA and protein in myocardial tissue of model group were significantly lower than the other four groups(P<0.05), whereas these levels in the AS-IV group were significantly higher than those in the other four groups (P<0.01). Additionally, the CID755673 group had significantly higher levels of PKD1, HDAC5 and VEGF mRNA and protein than the sham group, DMSO group and model group (P<0.05). CONCLUSION AS-IV may partly promote the angiogenesis of myocardial tissue in rats with myocardial infarction via the PKD1-HDAC5-VEGF pathway.
ABSTRACT
Objective To predict and verify the upstream regulatory microRNA (miRNA)of protein kinase D1 (PKD1),and to investigate its role in cerulein induced acute pancreatitis (AP)in rats. Methods Potential up-stream regulatory miRNA of PKD1 was predicted by using bioinformatics software. Dual luciferase reporter gene system and Western blot were applied to verify the regulation of PKD1 by the selected miRNA. Experimental AP was induced by 6 intraperitoneal injection of cerulein (20 μg/ kg)at hourly intervals after administration of the CY5 - labeled notar-get control (AP group,n = 20)or selected miRNA (treatment group,n = 20),respectively by intraperitoneal injection into rats. Other rats were divided randomly into a normal control group (n = 10)without any treatment. Besides 10 rats in either AP or treatment group were sacrificed 6 hours after the first injection of cerulein,and the rats were all sacri-ficed 24 hours after the first injection. The blood samples and pancreatic tissues of each rat were collected to test serum amylase and lipase activities,or to make hematoxylin - eosin stain for AP pathological scores as well as PKD1 immuno-histochemical staining,respectively. Results TargetScan 7. 1 software analysis showed that miR - 128 - 3p was the po-tential upstream regulatory miRNA of PKD1,which was verified by dual luciferase reporter gene system and Western blot detection. Compared to the normal control group,serum amylase and lipase activities after 6 h exposure to cerulein increased in both AP group and the treatment group[13313. 00(9424. 00 - 15995. 00)U/ L,13552. 00(10399. 50 -18408. 25)U/ L vs. 1430. 50(1214. 25 - 1543. 25)U/ L;547. 00 (515. 00 - 627. 00)U/ L,857. 50(522. 00 -1222. 25)U/ L vs. 34. 00(32. 50 - 34. 75)U/ L],and the differences were significant(χ2 = 8. 715,P < 0. 05;χ2 =9. 115,P < 0. 05),which indicated that the rat models of AP were successfully established. The immunohistochemical scores of PKD1 after 24 h exposure to cerulein decreased in the treatment group[0. 50(0 - 2. 75)scores],compared with the normal control group [4. 00(4. 00 - 8. 00)scores]and the AP group [4. 00(3. 75 - 8. 00)scores],and difference was significant(χ2 = 18. 302,P < 0. 05). Accordingly,the total pathological scores of HE staining decreased significantly in the treatment group,as compared to the AP group (3. 80 ± 0. 85 vs. 6. 90 ± 1. 15,t = 4. 481,P < 0. 01). The results showed that the inflammatory cell infiltration and tissue necrosis were significantly improved after miR -128 - 3p treatment. Conclusions miR - 128 - 3p is the upstream regulatory microRNA of PKD1 which protects pan-creata from necrotic injury and inflammatory cell infiltration in PKD1 - mediated acute pancreatitis.
ABSTRACT
Purpose To investigate the expression of protein kinase D1 (PKD1) in endometrial carcinoma and normal endometrium,and to investigate its relationship with the clinicopathological features of endometrial carcinoma.Methods Immunohistochemical SP and qRT-PCR was used to detect the mRNA and protein expression of PKD1 in 92 cases of endometrial cancer and 48 cases of normal endometrium,and to analyze the relationship of expression of PKD1 with the tumor differentiation and clinical stage of endometrial carcinoma tissue.Western blot method was used to detect the expression level of PKD1 protein in normal endometrial cell line and endometrial carcinorna cell lines with different degree of differentiation.Results mRNA and protein expression of PKD1 in endometrial carcinoma tissues was significantly higher than those in normal endometrial tissues (P ＜ 0.01),which showing a correlation to the degree of tissue differentiation and clinical pathologic staging.While,the expression level of PKD1 protein in endometrial cancer cell lines was also much higher than that in normal endometrial cells (P ＜ 0.01),and the lower differentiation,the higher level of PKD1 protein expression.Conclusion PKD1 is highly expressed in endometrial cancer patients.The level of PKD1 expression may be an important reference for predicting the malignant degree of endometrial cancer.
ABSTRACT
Objective To measure the expression of protein kinase D1 (PKD1),tyr463-phosphorylaed PKD1 (pPKD1-tyr463) and ser916-phos-phorylaed PKD1 (pPKD1-ser916) in squamous cell carcinoma (SCC),Bowen's disease (BD) and actinic keratosis (AK),and to explore their significance.Methods Fresh tissue samples were resected from lesions of patients with SCC (SCC group),BD (BD group) and AK (AK group),as well as from normal skin of healthy human controls (control group),and each group had a sample size of 10.Real-time RT-PCR was performed to measure the mRNA expression of protein kinase D1 gene (PRKD1),and Western blot analysis to determine the protein expression of PKD1,pPKD1-tyr463 and pPKD1-ser916.In addition,immunohistochemical study was conducted to determine the expression of PKD1,pPKD1-tyr463 and pPKD1-ser916 in another 50 paraffin-embedded skin samples of SCC,20 samples of BD,20 samples of AK and 10 normal skin samples.Results PRKD1 mRNA expression significantly differed among the control group (0.64 ± 0.09),SCC group (5.37 ± 1.06),BD group (2.69 ± 0.72) and AK group (2.43 ± 0.46) (F =21.37,P ＜ 0.05),and was significantly higher in the SCC,BD and AK groups than that in the control group (P ＜ 0.05),as well as in the SCC group than that in the AK and BD groups (both P ＜ 0.05).However,no significant difference in the PRKD1 mRNA expression was observed between the BD group and AK group (P ＞ 0.05).Immunohistochemical study showed that the total PKD1 protein and pPKD1-tyr463 in the SCC and BD groups were mainly expressed in the cytoplasm and cell membrane of spinous layer cells and atypical cells,and their expression rates were significantly higher than those in the AK group and control group (all P ＜ 0.01).The pPKD1-ser916 was only slightly expressed in some cancer nests of well-differentiated SCC tissues,but not in poorly-differentiated SCC,AK,BD tissues and normal skin tissues.In the SCC group,the expression rate of PKD1 increased with the increase of the pathological grade of SCC,and the PKD1 expression was positively correlated with pPKD1-tyr463 expression (rcc =0.479,P ＜ 0.05).Western blot results were consistent with immunohistochemical findings.Conclusion PKD1 and pPKD1-tyr463 may be involved in the development and differentiation of skin tumors derived from stratified squamous epithelium,and PKD1 may exert promotive effects on the formation of cutaneous SCC by activating the Tyr463 phosphorylation site.
ABSTRACT
Aim To explore the effect of protein kinase D1 (PKD1 )on adherence,migration,proliferation, microvascular formation,and eNOS expression in bone marrow-derived endothelial progenitor cells(EPCs)of rats.Method The EPCs were isolated from bone marrow of rats,cultured and detected,the effects of PKD1 and its specific blocking agent CID755673 on adhesion,migration,proliferation,or microvascular formation were observed,as well as mRNA and protein expression of endothelial nitric oxide synthase (eNOS ) in EPCs.Results PKD1 significantly promoted adhe-sion,migration,proliferation and microvascular forma-tion of EPCs,and upregulated the mRNA and protein expression of eNOS in EPCs,according to the cell cul-ture experiments of EPCs in vitro.Conclusion PKD1 has the role of angiogenesis through regulation of EPCs,which might be dependent on eNOS.