Tinopanoids K-T, clerodane diterpenoids with anti-inflammatory activity from Tinospora crispa.

A total of 17 structurally diverse clerodane diterpenoids, including ten undescribed clerodane diterpenoids (tinopanoids K-T, 1-10) and seven known compounds (11-17), were isolated from the vines and leaves of Tinospora crispa. Compound 3 has not only bear the dominant substituents of γ-hydroxy-α, ß-unsaturated-γ-lactone with anti-inflammatory activity, but also a ternary epoxy structure at C-3/C-4. The planar structures and relative configurations of the clerodane diterpenoids were elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1, 4, 8 and 13 were determined by single-crystal X-ray crystallographic, while that of compound 3 was determined using computed ECD data and single crystal X-ray diffraction of related p-bromobenzoate ester (3a). Subsequently, all compounds were evaluated for their inhibitory effect on nitric oxide (NO) production of LPS-activated BV-2 cells, and compounds 3 and 8 exhibited better NO inhibitory potency, with IC50 values of 5.6 and 13.8 µM than the positive control minocycline (Mino, IC50 = 22.9 µM). The corresponding results of western blot analysis and qRT-PCR revealed that compound 3 can significantly inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions, mRNA levels of pro-inflammatory cytokins of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß). The underlying mechanism by which compound 3 exerted anti-neuroinflammatory effects was investigated by western blot and immunofluorescence assay, which suggested compound 3 inhibited LPS induced neuroinflammation via the suppression of toll-like receptor 4 (TLR4) dependent Signal Transducer and Activator of Transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of Heme Oxygenase-1 (HO-1) mediated signals.

[Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene].

OBJECTIVE: To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene. METHODS: Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 DNA ligase ligation. Based on the human mda7 gene sequences, PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro. After the candidate clones were identified by DNA sequencing, the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles. Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector. The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot, respectively. The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively. RESULTS: ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector. The transfection efficiency of the collected virus exceeded 90% in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency. The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly. The mda7-pLVX-Puro lentiviral vector increased the expression of mda7 in PC-3 cells, and the ability was maintained for one month. Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells (Ps<0.05). CONCLUSION: The lentiviral vectors of ILK knockdown and mda7 over-expression have been successfully constructed and identified. The recombinant lentivirus can efficiently infect human prostate cancer PC-3 cells, in which ILK expression is inhibited and mda7 is over-expressed.

Clerodane diterpenoids with in-vitro anti-neuroinflammatory activity from the tuberous root of Tinospora sagittata (Menispermaceae).

Twenty-six clerodane diterpenoids have been isolated from T. sagittata, a plant species of traditional Chinese medicine Radix Tinosporae, also named as "Jin Guo Lan". Among them, there are eight previously undescribed clerodane diterpenoids (tinotanoids A-H: 1-8), and 18 known diterpenoids (9-26). The absolute configurations of compounds 1, 2, 5, 8, 13, 17 and 20 were determined by single-crystal X-ray diffraction. Compound 1 is the first example of rotameric clerodane diterpenoid with a γ-lactone ring which is constructed between C-11 and C-17; meanwhile, compounds 3 and 4 are two pairs of inseparable epimers. Compounds 2, 12 and 17 demonstrated excellent inhibitory activity on NO production against LPS-stimulated BV-2 cells with IC50 values of 9.56 ± 0.69, 9.11 ± 0.53 and 11.12 ± 0.70 µM, respectively. These activities were significantly higher than that of the positive control minocycline (IC50 = 23.57 ± 0.92 µM). Moreover, compounds 2, 12 and 17 dramatically reduced the LPS-induced upregulation of iNOS and COX-2 expression. Compounds 2 and 12 significantly inhibited the levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 that were increased by LPS stimulation.

New polychlorinated bibenzyls from Rhododendron minutiflorum.

Five new polychlorinated bibenzyls (1-5) along with 3 known compounds (6-8) were isolated from the stems and leaves of Rhododendron minutiflorum. The chemical structures of all the isolates were determined by spectroscopic methods, and compounds 1 and 2 were further verified by single-crystal X-ray diffraction analyses. Compounds 1-5 were halogenated compounds which bear three to five chlorine atoms in their chemical structures. Biologically, compounds 2, 5 and 6 showed varying degrees of toxicity toward the Asian citrus psyllid (Diaphorina citri) with LD50 values 27.15, 17.02 and 16.20 mg/L, respectively. These values were comparable to the positive control matrine (LD50 = 11.86 mg/L), which were calculated using observations on day 6. Meanwhile, compound 4 had α-glucosidase inhibitory activity with IC50 value of 17.87 ± 0.74 µM.

Terpenoids with α-glucosidase inhibitory activity from Rhododendron minutiflorum Hu.

Five undescribed triterpenoids, two unusual omphalane-type sesquiterpenoids together with twenty-five known compounds were isolated from the leaves and stems of Rhododendron minutiflorum Hu. The absolute configurations of 1-3 and 6 were established by single-crystal X-ray diffraction analysis and electronic circular dichroism (ECD). Compounds 6-7 feature the rare omphalane-type sesquiterpene skeleton and are verified by single-crystal X-ray diffraction analysis for the first time. In the biological activity assay, most of the triterpenoids have different degrees of inhibitory effects on α-glucosidase, with IC50 values ranging from 6.97 to 229.3 µM (the positive control drug acarbose has an IC50 value of 3.07 × 10-3 µM). Structure and activity relationship (SAR) study reveals that the oxidation degrees of C-3, C-8, or C-11 to C-13 of the ursane-type triterpenoid influence the inhibitory activity dramatically.

[Clinicopathologic study of parvovirus B19 infection in perinatal period].

OBJECTIVE: To characterize the risks and histopathological features of parvovirus B19 infection of infants in perinatal period. METHODS: Routine pathological examination was performed on 1 neonate, 2 dead fetuses and 2 placentas using either autopsy or biopsy materials. RESULTS: The diagnostic intranuclear inclusions were found in erythroblasts in the bone marrow, liver, spleen and lungs in one case, in the spleen and liver in one case, in the spleen in one case, and in the placentas in two cases. CONCLUSIONS: Severe hemolytic anemia or fetal hydrop or hemophagocytosis caused by the infection of parvovirus B19 can lead to death of infected neonates and fetus. Pathological confirmation of parvovirus B19 infection relies on the identification of erythroblasts containing the diagnostic intranuclear inclusions.

[Pseudoangiosarcomatous squamous cell carcinoma of the penis: a case report with clinicopathological and human papilloma virus analyses].

OBJECTIVE: To further understand the clinicopathological, ultrastructural and molecular features of penile pseudoangiosarcomatous squamous cell carcinoma (PASCC), and improve its diagnosis and treatment. METHODS: A 47-year-old male patient with penile PASCC was reported and the relevant literature reviewed. The main clinical manifestations of the patient were a typical surface ulceration with hemorrhage and purulent secretion with a foul smell, a papillary mass about 5.0 cm x 5.0 cm x 4.0 cm for 1 year on the foreskin of the penis, and 3 enlarged bilateral inguinal lymph nodes. CT scanning showed no enlarged lymph nodes in the abdomen and pelvis, and X-ray examination revealed no abnormality in the chest. RESULTS: The diagnosis was established by biopsy. Partial penectomy and bilateral inguinal lymphadenectomy (T2N2M0) were performed, followed by adjuvant pelvic radiotherapy. Two months later, total penectomy was necessitated by penile flap necrosis and local recurrence. Eleven months after the first surgery, the patient died of extensive metastasis to the pelvis and lungs. Under the light microsope, the tumor was mainly composed of vessel-like lacunar reticularis spindle cells and a few local squamous cancer cells. Careful examination revealed some focal areas with evident transition from squamous nests to the more acantholytic areas extending towards the pseudoangiosarcomatous spaces. Pathogenetically, it appeared to be the variant of acantholytic squamous cell carcinoma. Immunohistochemically, most tumor cells were strongly positive for keratin (AE1/AE3) and focally positive for EMA, with the typical squamous cells focally positive for 34betaE12 and vimentin. The vessels that proliferated in the tumor were decorated by CD31, CD34 and factor VIII-related antigens, but the tumor cells were negative for HMB45, SMA, Desmin and CEA. HPV DNA (HPVpan, HPV6B/11, HPV16/18, HPV31/33) was not detected by in situ hybridization in the primary and metastatic tumors. CONCLUSION: PASCC is a specific and extremely rare subtype of penile SCC with dramatic similarity to angiosarcoma under the microscope, with poor prognosis. Its diagnosis depends on histopathological, immunohistochemical and ultrastructural studies. Such a presentation underscores the importance of timely consultation, early diagnosis and prompt treatment.

[Perivascular epithelioid cell tumor of uterus: report of 5 cases and literature review].

OBJECTIVE: To study the pathologic features, diagnosis, differential diagnosis and biologic behavior of uterine perivascular epithelioid tumor. METHODS: Five cases of uterine perivascular epithelioid cell tumor were studied by light microscopy and immunohistochemistry. Follow-up information was reviewed. RESULTS: All the five tumors were composed by clear or eosinophilic cells arranged in nests and cords, associated with abundant small vessels and hyalinization in the stroma. Immunohistochemically, the tumor cells demonstrated positive staining for melanocytic markers (HMB45 and/or Melan-A), desmin and smooth muscle actin. The staining for cytokeratin and CD10 was negative. All the patients followed for a certain period are still alive, with no evidence of disease recurrence. CONCLUSIONS: Perivascular epithelioid cell tumor is a rare mesenchymal tumor of uterus, with distinctive histologic and immunohistochemical features. It should be distinguished from clear cell carcinoma and epithelioid leiomyoma of uterus. Positivity for melanocytic markers (especially HMB45) plays an important role in the diagnosis of this tumor. In general, the tumor is categorized as benign, with uncertain malignant potential and malignant.