Research on the anti-tumor effect of nordihydroguaiaretic acid targeting MyD88 / 药学学报

This study mainly explores the role of myeloid differentiation primary response protein 88 (MyD88) in tumorigenesis and development, to identify active compounds targeting MyD88. CRISPR/Cas9 system and xenograft tumor model were used to detect the effect of MyD88 deletion on tumor growth, and the experimental animal ethics review number was PZSHUTCM200828006. Microscale thermophoresis technology (MST) was used to identify compounds directly bind to MyD88 and further detect the impact of candidate small molecules on cell proliferation. Results showed that depletion of MyD88 significantly inhibited xenograft tumor growth of colon cancer, pancreatic cancer and skin cancer and the activity of NF-κB signaling pathway. MST showed that nordihydroguaiaretic acid (NDGA) bound to MyD88, with the binding dissociation constant Kd of 14.61 µmol·L-1. NDGA inhibited NF-κB reporting system activation and phosphorylation of p65, the key factor in NF-κB signal pathway. In addition, the results of colony formation assay showed that NDGA suppressed the proliferation of tumor cells. The above results show that, MyD88 is a potential therapeutic target for colon cancer, pancreatic cancer and skin cancer, NDGA directly binds to MyD88 and inhibits the activity of NF-κB signaling pathway, as well as inhibits the proliferation of pancreatic cancer, skin cancer and colon cancer cells.

Nordihydroguaiaretic acid reverses efflux pump system MexCD-OprJ mediated ceftazidime resistance of pseudomonas aeruginosa and its related mechanisms / 中草药

Objective: To evaluate the effect of nordihydroguaiaretic acid (NDGA) on ceftazidime resistance of Pseudomonas aeruginosa mediated by efflux pump system MexCD-OprJ and explore its mechanism. Methods: The bacterial solution with a concentration of 0.5 mcburney was diluted and inoculated in a 96-well plate, and NDGA and ceftazidime were added by the checkerboard dilution method. At the same time, the untreated control group, NDGA control group and ceftazidime control group were set; After being cultured for 24 h, the absorbance was measured by an enzyme micro-plate reader, the minimum inhibitory concentration (MIC) of each drug was recorded and the bacteriostatic rate and fractional inhibitory concentration (FIC) index were calculated. Bacteria were inoculated with the bacterial liquid coating method in the 96-well plates, and the bacterial colony number was counted after 24 h of culture. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the gene expressions of efflux pump membrane protein MexC, MexD, OprJ and nfxB. Results: Compared with ceftazidime or NDGA alone, combination of ceftazidime and NDGA significantly inhibited the growth of efflux pump system MexCD-OprJ-mediated ceftazidime-resistant P. aeruginosa (P < 0.05); The pharmacological effects of ceftazidime and NDGA showed synergistic or additive effects; After combined administration, the MIC values of ceftazidime and NDGA were significantly decreased, and the MIC value of some ceftazidime had no significant difference from that of ceftazidime-sensitive P. aeruginosa; Compared with ceftazidime alone, the gene expressions of efflux pump membrane proteins MexC, MexD and OprJ were significantly decreased after combined application of ceftazidime and NDGA (P < 0.05), while the expression of nfxB was significantly increased (P < 0.05). Conclusion: The mechanism of NDGA on ceftazidime resistance of P. aeruginosa mediated by efflux pump system MexCD-OprJ is related to its ability to down-regulate the gene expression of efflux pump membrane proteins MexC, MexD and OprJ, and up-regulate the gene expression of the negative regulatory gene nfxB of the above three proteins.

Effects of Lipoxygenase Inhibitor on Diabetic Nephropathy in Rats: Decreasing Proteinuria and Preserving Renal Function / 대한신장학회지

PURPOSE: Oxidative stress leads to an increased production of lipoxygenase derivatives in diabetic nephropathy. Thus, we hypothesized that lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), ha the effects of decreasing proteinuria and preserving renal function in streptozotocin (STZ)-induced diabetic rats. METHODS: 45 Sprague-Dawley rats were divided into three groups; (A) treatment with lipoxygenase inhibitor, NDGA in diabetic nephropathy rats, (B) treatment with dimethyl sulfoxide (DMSO) as a vehicle in STZ-induced diabetic rats, (C) normal control group with subcutaneous injection of normal saline. Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg) in rats of group A and B. After the 4th week of STZ injection, NDGA (10 mg/kg) and DMSO were given subcutaneously for another 4 weeks in group A and B respectively. RESULTS: The NDGA-treated diabetic rats exhibited significantly decreased urinary albumin excretion. Serum creatinine and blood urea nitrogen concentrations were increased in both group A and B, and tend to be higher in group B than group A. Twenty-four-hour urine creatinine clearances were increased in both group A and B after injection of STZ. Pathologic alterations of kidney were observed after injection of STZ, and then attenuated after administration of NDGA. CONCLUSION: These results suggest the potential of lipoxygenase inhibitor as a complementary therapy for the prevention and treatment of diabetic nephropathy.

Research on apoptosis of human colon carcinoma cell induced by NDGA combined Celecoxib / 中国医师杂志

Objective To study the effect of 5-lipoxygenase(5-LOX) inhibitor nordihyroguaiaretic acid (NDGA) combined the selective cyclooxygenase-2 (COX-2) inhibitor Celecoxib on the apoptosis of human colon carcinoma cell line HT-29. Methods Different concentration of NDGA and Celecoxib combinations were used to process cancer cell, and thiazolyl blue tetrazlium bromide (MTT) and phase contrast microscope and Annexin V/PI fluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to study the proliferation inhibited effect and apoptosis induced effect caused by combination of NDGA combined Celecoxib. Results MTT results showed that the viability of NDGA group, Celecoxib group and the group of NDGA combined Celecoxib (0.432±0.024,0.425±0.013,0.303±0.014 vs 0.693±0.018,t＝18.79,25.75,37.64,P＜0.01) was obviously lower than control group. The group of NDGA combined Celecoxib was significantly lower than NDGA group or Celecoxib group (t＝10.21, 14.14,P＜0.01). Under inverted phase contrast microscope, cell morphology significantly changed, and the group of NDGA combined Celecoxib changed most obviously. Apoptosis was observed by laser scanning confocal microscope (LSM) after NDGA and Celecoxib were used to process the HT-29. RT-PCR showed that up-regulation of Caspase-3 after treatment, and the combination of two drugs increased the most. Conclusions NDGA combined Celecoxib inhibited proliferation and induced apoptosis in human colon carcinoma cell line HT-29, and combined therapy had better effect than that of any drug used separate-ly. The mechanism may be associated with up-regulation of Caspase-3.

Effect of nordihydroguaiaretic acid on chronic atrophic gastritis in rats / 中国医师杂志

Objective To study the preventive effect of lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on chronic atrophic gastritis (CAG). Methods To construct CAG rat model, 72 male rats were randomly divided into 6 groups, including normal group, CAG model group, NDGA group with different dose and positive groups (folacin). After feeding for 24 weeks, all rats were executed, their stomach mucous membrane was picked out and stained with H.E. The expression of 5-LOX and P16 protein in mucous membrane epithelia cells were detected by immunohistochemistry. Results The CAG incidence rate in model group was significant higher than normal group (77.8% vs 0% , P ＜0. 05), which indicated that C AG model rat was successfully established. The CAG incidence rate of model group was significantly higher than NDGA groups (77.8% vs 25% ,27.3% ,25%, P＜0.05), while no significant difference was found between positive group and NDGA groups (30% vs 25% ,27.3% ,25% , P＞0.05) .The expression of 5-lox in model group was higher than NDGA groups (44% vs 25% ,27% ,25%, P＜0.05). The expression of P16 protein in model group was lower than NDGA groups (66.7% vs 83.3% ,81.8% ,83.3%,P＜0.05) , and there were no significant differences between NDGA groups and positive group (83. 3%,81.8% ,83.3% vs 80%, P＞0.05). Conclusions NDGA could prevent the occurrence of N-ethyl-N-nitro-N nitrosoguanidine-induced chronic atrophic gastritis in rat. NDGA could down-regulate the expression of 5-LOX and up-regulate the expression of P16 in stomach mucous membrane of N-ethyl-N-nitro -N-nitrosoguanidine-induced chronic atrophic gastritis in rat.

Post-treatment Effects of Erythropoietin and Nordihydroguaiaretic Acid on Recovery from Cisplatin-induced Acute Renal Failure in the Rat

5-Lipoxygenase inhibitor and human recombinant erythropoietin might accelerate renal recovery in cisplatin-induced acute renal failure rats. Male Sprague-Dawley rats were randomized into four groups: 1) normal controls; 2) Cisplatin group-cisplatin induced acute renal failure (ARF) plus vehicle treatment; 3) Cisplatin+nordihydroguaiaretic acid (NDGA) group-cisplatin induced ARF plus 5-lipoxygenase inhibitor treatment; 4) Cisplatin+erythropoietin (EPO) group-cisplatin induced ARF plus erythropoietin treatment. On day 10 (after 7 daily injections of NDGA or EPO), urea nitrogen and serum Cr concentrations were significantly lower in the Cisplatin+NDGA and Cisplatin+EPO groups than in the Cisplatin group, and 24 hr urine Cr clearances were significantly higher in the Cisplatin+EPO group than in the Cisplatin group. Semiquantitative assessments of histological lesions did not produce any significant differences between the three treatment groups. Numbers of PCNA(+) cells were significantly higher in Cisplatin, Cisplatin+NDGA, and Cisplatin+EPO groups than in normal controls. Those PCNA(+) cells were significantly increased in Cisplatin+NDGA group. These results suggest that EPO and also NDGA accelerate renal function recovery by stimulating tubular epithelial cell regeneration.

Effects of Celecoxib and Nordihydroguaiaretic Acid on Puromycin Aminonucleoside-Induced Nephrosis in the Rat

The selective cyclooxygenase-2 (COX-2) and 5-lipoxygenase (LOX) inhibitors might inhibit prostaglandin synthesis and reduce proteinuria. The present study was designed to investigate the anti-proteinuric effects of nordihydroguaiaretic acid (NDGA) as compared with celecoxib in puromycin aminonucleoside (PAN) nephrosis rats. Fifty five male Sprague-Dawley rats were divided into 4 groups; A, normal control; B, PAN group; C, PAN+COX-2 inhibitor (celecoxib) group; and D, PAN+5-LOX inhibitor (NDGA) group. After induction of PAN nephrosis through repeated injections of PAN (7.5 and 15 mg/100 g body weight), rats were treated with celecoxib, NDGA, or vehicle for 2 weeks. Twenty four hour urine protein excretions were significantly lower in PAN+celecoxib and PAN+NDGA groups than in PAN group. Serum creatinine (SCr) concentrations and 24 hr urine creatinine clearances (CCr) were not significantly different in the four groups. Electron microscopy showed that podocyte morphology was changed after the induction of PAN nephrosis and was recovered after celecoxib or NDGA administration. Celecoxib significantly recovered the expressions of nephrin, CD2AP, COX-2, and TGF-beta. NDGA also recovered TGF-betaexpression, but did not alter the expressions of nephrin, CD2AP and COX-2. The present study suggested that celecoxib and NDGA might effectively reduce proteinuria in nephrotic syndrome without impairing renal function.

Effects of Nordy on proliferation, apoptosis, and differentiation of HPV 16 subgenes-immortalized human endocervical cells / 肿瘤

Objective: To investigate the effects of Nordy on proliferation, differentiation, and apoptosis of HPV 16 subgenes (E6,E7)-immortalized human endocervical cells (H8 cells). Methods: The inhibitory effects of Nordy on proliferation of H8 cells were measured by MTT assay. The expression of nuclear antigen mcm 5 in H8 cells was detected by immunocytochemical SP method. The effect of Nordy on cell cycle and apoptosis of H8 cells was analyzed by flow cytometry (FCM). Morphological changes of H8 cells were observed by light and electron microscopy. The activity of telomerase was tested by telomeric repeat amplification protocal-enzyme linked immunosorbent assay (TRAP-ELISA). Results: Nordy 10-100 μmol/L inhibited the proliferation of H8 cells to different extent, decreased the intracellular expression of mcm 5 protein, and arrested H8 cells in G0/G1 phase, reduced the proportion of H8 cells in S phase, and increased the apoptotic rate. Morphological examination showed that Nordy-treated H8 cells tended to differentiate into mature cells. The activity of telomerase decreased significantly after Nordy treatment. Conclusion: Nordy inhibites proliferation, activates apoptosis, and induces differentiation of H8 cells by blocking cell cycle and decreasing activity of telomerase.

Effects of nordihydroguaiaretic acid on the endometriosis in Bama miniature pigs / 第三军医大学学报

Objective To investigate the effects of nordihydroguaiaretic acid(NDGA)on the endometriosis established in Bama miniature pigs.Methods Six Bama pigs that had been successfully established into animal model of endometriosis subcutaneously received 3% NDGA at dose of 20 mg/kg(n=4)or PBS solution(n=2)for 20 d.The grafts in abdominal cavity and subcutaneous tissues were observed and the serum concentrations of hormone,were measured by chemiluminoimmunoassay before and after the period of NDGA or PBS injection.The expressions of FⅧAg and VEGF in the endometriotic tissues were observed.Results The endometriotic masses were reduced to some degree after the time period of NDGA injection,even partially disappeared,while those treated with PBS showed no obvious changes.Histoimmunochemical analysis showed NDGA decreased the number of endometrial glands,the proliferation rate of the glandular epithelial cells,the endometrial stroma cells and the vessels,and inhibited the microvascular density and VEGF in the endometriotic tissues(P

Effects of NDGA on the growth of a human malignant glioma cell line CHG-5 / 第三军医大学学报

Objective To investigate the effects of nordihydroguaiaretic acid (NDGA) on the growth of a human malignant glioma cell line CHG 5 in vitro and in vivo . Methods Colorimetric MTT assay, flow cytometry, and light microscopy were used to investigate the proliferation in vitro , cell cycles, apoptosis of CHG 5 cells, and the growth of xenografted tumor in nude mice. Results NDGA significantly inhibited the proliferation of CHG 5 cells in vitro . Cells in G 0/G 1 phase increased, but cells in S, G 2/M phases decreased, and apoptotic cells increased significantly. After treatment of NDGA (50 mg/kg, intraperitoneally) at 5 d after the inoculation of tumor cells, the xenografted tumor volume reduced remarkably without causing significant toxic and side effects. Conclusion The inhibitory effect of NDGA on the growth of CHG 5 cells may be correlated with the regulation of cell cycles and induction of apoptosis.

Effects of nordihydroguaiaretic acid on early embryo in rats / 第三军医大学学报

Objective To investigate the effects of nordihydroguaiaretic acid (NDGA) on the embryos growth and decidual development in rats. Methods Fifty pregnant SD rats were randomly divided into NDGA-treated group(N)and control group (P), which were given subcutaneous injection of NDGA or PBS on embryonic day 1(E1), E7 and E14, and then killed on E6 and E9. The number of implantations was calculated, the diameter of the embryos was measured and the numbers and weight of neonates were recorded. The expressions of VEGF, PCNA and FⅧRAg in the deciduas and embryos were detected by immunohistochemistry and then made image analysis. The serum concentrations of progesterone on E9 were measured. Results The number of implantations were decreased with NDGA treatment on E1 (P

Morphological changes and differential protein spectrum of human malignant glioma cell SHG-44 after treated with Nordy,an analog of Nordihydroguaiaretic acid / 第二军医大学学报

Objective:To observe the morphological changes and analyze the differential protein spectrum of human malignant glioma cells SHG-44 after treated with Nordy(Chinese patent number:ZL02133700.4),an analog of Nordihydroguaiaretic acid.Methods: The differentiation of SHG-44 cells was induced by 100 ?mol/L or 200 ?mol/L Nordy;the morphological changes of cells were observed 24,48 and 72 h after Nordy treatment and the findings were compared with those of the control group(received no treatment).The total proteins were extracted from SHG-44 cells treated with 200 ?mol/L Nordy for 72 h and cells in control group,then were subjected to two-dimensional gel electrophoresis.PDquest 7.1 software was employed to compare the protein expression differences.The highly expressed differential proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS).Results: The morphological changes of SHG-44 cells treated with 200 ?mol/L Nordy were more obvious than those treated with 100 ?mol/L Nordy,and the most obvious differentiation was found in the cells treated for 72 h.Compared with those of control group,23 differential protein spots were identified by the two-dimensional electrophoresis,including 21 down-regulated ones and 2 up-regulated ones.MALDI-TOF-MS showed that the highly expressed proteins were: an unknown protein,proliferation-associated gene A,Up1,alternative splicing factor ASF-3,cofilin1(non-muscle),eukaryotic translation initiation factor 5A,beta galactoside binding lectin,and glutathione-S-transferase Pi.Conclusion: Nordy can induce differentiation of human malignant glioma cells SHG-44 in a time-effect and dose-effect dependent manner.The Nordy-induced differential proteins may function in multiple aspects such as cell proliferation,apoptosis and gene transcription.