Pretreatment with atorvastatin ameliorates cobra venom factor-induced acute lung inflammation in mice.

BACKGROUND: The complement system plays a critical role as the pathogenic factor in the models of acute lung injury due to various causes. Cobra venom factor (CVF) is a commonly used complement research tool. The CVF can cause acute inflammation in the lung by producing complement activation components. Atorvastatin (ATR) is a 3-hydroxy-3-methylglutaryl coenzyme A inhibitor approved for control of plasma cholesterol levels. This inhibitor can reduce the acute pulmonary inflammatory response. However, the ability of ATR in treating acute lung inflammation caused by complement activation is still unknown. Therefore, we investigated the effect of ATR on lung inflammation in mice induced by activation of the complement alternative pathway in this study. METHODS: ATR (10 mg/kg/day via oral gavage) was administered for 7 days before tail vein injection of CVF (25 µg/kg). On the seventh day, all mice were sacrificed 1 h after injection. The lung lobe, bronchoalveolar lavage fluid (BALF), and blood samples were collected. The myeloperoxidase (MPO) activity of the lung homogenate, the leukocyte cell count, and the protein content of BALF were measured. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), P-selectin, and Intercellular cell adhesion molecule-1 (ICAM-1) in BALF and serum were determined by enzyme-linked immunosorbent assay. The pathological change of the lung tissue was observed by hematoxylin and eosin staining. The deposition of C5b-9 in the lung tissue was detected by immunohistochemistry. The phosphorylation of NF-κB p65 in the lung tissues was examined by immunohistochemistry and western blotting. RESULTS: The lung inflammation levels were determined by measuring the leukocyte cell numbers and protein content of BALF, the lung MPO activity, and expression and staining of the inflammatory mediators (IL-6 and TNF-α), and adhesion molecules (P-selectin and ICAM-1) for lung lesion. A significant reduction in the lung inflammation levels was observed after 7 days in ATR pre-treated mice with a CVF-induced lung disease. Deposition of C5b-9 was significantly alleviated by ATR pretreatment. Early intervention with ATR significantly reduced the development of acute lung inflammation on the basis of phosphorylation of NF-κB p65 in the lung. CONCLUSION: These findings suggest the identification of ATR treatment for the lung inflammation induced by activating the complement system on the basis of its anti-inflammatory response. Together with the model replicating the complement activating characteristics of acute lung injury, the results may be translatable to the overactivated complement relevant diseases.

Complement C3 participates in the function and mechanism of traumatic brain injury at simulated high altitude.

BACKGROUND: Traumatic brain injury (TBI) leads to severe mortality and disability, in which secondary injury induced by complement activation plays an important role. TBI tends to be associated with more severe cerebral edema and worse neurological functional recovery if it occurs in high-altitude areas than in low-altitude areas. However, the underlying mechanism of this difference is unknown. Thus, we used cobra venom factor (CVF) to deplete complement C3 in simulated high-altitude areas to explore whether the differences in outcome at different altitudes are related to secondary injury caused by complement C3. METHODS: The weight-drop model was adopted to induce TBI in rats. Rats were randomly divided into the following groups: sham + saline (sham), high altitude + TBI + saline (HAT), high altitude + TBI + CVF (H-CVF), low altitude + TBI + saline (LAT), and low altitude + TBI + CVF (L-CVF). Brain contusion and edema volumes, brain water content, myelin basic protein (MBP) expression, tumor necrosis factor alpha (TNF-a) expression, interleukin 1 beta (IL1B) expression, mortality rate, neurological function, and complement component 3 (C3) mRNA expression were measured by techniques such as Evans blue fluorescence, Perls staining, TUNEL staining, ELISA, immunohistochemistry and Western blotting to evaluate correlations between complement activation and secondary injury. RESULTS: The activation of complement after TBI was significantly higher at high altitude than at low altitude. High-altitude TBI resulted in a leakier blood-brain barrier, more severe cerebral edema and higher mortality than low-altitude TBI did. In addition, high-altitude TBI tended to be associated with more MBP degradation, ferric iron deposition, neuronal apoptosis, and inflammatory factor deposition than low-altitude TBI. All of these effects of TBI were partially reversed by inhibiting complement activation using CVF. CONCLUSION: Our study provided evidence that TBI at high altitude leads to severe edema and high mortality and disability rates. Complement C3 activation is one of the important factors contributing to secondary brain injury.

Effects of systemic inhibitors of acid-sensing ion channels 1 (ASIC1) against acute and chronic mechanical allodynia in a rodent model of migraine.

BACKGROUND AND PURPOSE: Acid-sensing ion channels (ASICs) are neuronal proton sensors emerging as potential therapeutic targets in pain of the orofacial region. Amiloride, a non-specific ASIC blocker, has been shown to exert beneficial effects in animal models of migraine and in patients. We explored the involvement of the ASIC1-subtype in cutaneous allodynia, a hallmark of migraine affecting cephalic and extra-cephalic regions in about 70% of migrainers. EXPERIMENTAL APPROACH: We investigated the effects of systemic injections of amiloride and mambalgin-1, a specific inhibitor of ASIC1a- and ASIC1b-containing channels, on cephalic and extra-cephalic mechanical sensitivity in a rodent model of acute and chronic migraine induced by i.p. injections of isosorbide dinitrate. KEY RESULTS: I.v. injections of these inhibitors reversed cephalic and extra-cephalic acute cutaneous mechanical allodynia in rats, a single injection inducing a delay in the subsequent establishment of chronic allodynia. Both mambalgin-1 and amiloride also reversed established chronic allodynia. The anti-allodynic effects of mambalgin-1 were not altered in ASIC1a-knockout mice, showing the ASIC1a subtype is not involved in these effects which were comparable to those of the anti-migraine drug sumatriptan and of the preventive drug topiramate on acute and chronic allodynia respectively. A single daily injection of mambalgin-1 also had a significant preventive effect on allodynia chronification. CONCLUSIONS AND IMPLICATIONS: These pharmacological data support the involvement of peripheral ASIC1-containing channels in migraine cutaneous allodynia as well as in its chronification. They highlight the therapeutic potential of ASIC1 inhibitors as both an acute and prophylactic treatment for migraine.

Comparative Efficacy of Drugs for Preventing Acute Kidney Injury after Cardiac Surgery: A Network Meta-Analysis.

BACKGROUND: Acute kidney injury (AKI) occurs frequently after cardiac surgery and has been associated with increased hospital length of stay, mortality, and costs. OBJECTIVE: We aimed to evaluate the efficacy of pharmacologic strategies for preventing AKI after cardiac surgery. METHODS: We searched PubMed, Embase, the Cochrane Central Register of Controlled Trials (CENTRAL) up to 6 May 2017 and the reference lists of relevant articles about trials. The outcome was the occurrence of AKI. This is the first network meta-analysis of the different prevention strategies using Bayesian methodology. RESULTS: The study included 63 articles with 19,520 participants and evaluated the effect of ten pharmacologic strategies to prevent AKI in patients undergoing cardiac surgery. Compared with placebo, the odds ratio (OR) for the occurrence of AKI was 0.24 [95% confidence interval (CI) 0.16-0.34] with natriuretic peptide, 0.33 (95% CI 0.14-0.70) with fenoldopam, 0.54 (95% CI 0.31-0.84) with dexmedetomidine, 0.56 (95% CI 0.29-0.95) with low-dose erythropoietin, 0.63 (95% CI 0.43-0.88) with levosimendan, 0.76 (95% CI 0.52-1.10) with steroids, 0.83 (95% CI 0.48-1.40) with high-dose erythropoietin, 0.85 (95% CI 0.64-1.14) with N-acetylcysteine, 0.96 (95% CI 0.69-1.29) with sodium bicarbonate, and 1.05 (95% CI 0.70-1.41) with statins. The surface under the cumulative ranking curve probabilities indicated that natriuretic peptide was the best treatment therapy and that fenoldopam ranked second. CONCLUSIONS: Natriuretic peptide is probably the preferred pharmacologic strategy to prevent AKI in adult patients undergoing cardiac surgery, especially in those at high risk of AKI.

Suppression of Ehrlich carcinoma growth by cobra venom factor.

Cobra venom factor (CVF) depletes the complement system of the blood by forming stable convertase C3/C5 of the alternative pathway. We found that CVF from the Thailand cobra venom slows down the growth of subcutaneous Ehrlich carcinoma (EC) in mice at a dose of 1.7 nmol/g. Previously, we described a similar effect for the nerve growth factor (NGF) from the venom of this cobra. However, these factors did not exhibit either synergy or additive effect. On the contrary, they neutralized the antitumor effect of each other when they were administered simultaneously. Therefore, on the one hand, the NGF antitumor effect against EC manifests itself under the conditions of inflammation, and normal functioning of the complement system is necessary for this effect to occur. On the other hand, suppression of the humoral immune system leads to a slowdown of the EC growth, but administration of NGF prevents this.

Protection against osteoarthritis in experimental animals by nanogold conjugated snake venom protein toxin gold nanoparticle-Naja kaouthia cytotoxin 1.

BACKGROUND & OBJECTIVES: Increased severity of osteoarthritis (OA) and adverse side effects of its treatment led to the search for alternative therapies. It was previously reported that snake venom protein toxin Naja kaouthia cytotoxin 1 (NKCT1) and gold nanoparticle (GNP) individually have potential against excremental arthritis. In this study, we analyzed the protective activity of GNP conjugated protein toxin NKCT1 (GNP-NKCT1) against experimental OA. METHODS: Gold nanoparticle conjugation with NKCT1 (GNP-NKCT1) was done and its physiochemical properties were studied. OA was induced in male albino rats by intra-articular injection of bacterial collagenase and treatment was done with NKCT1/GNP-NKCT1/standard drug (indomethacin). Physical parameter (ankle diameter), urinary markers (hydroxyproline, glucosamine, pyridinoline, deoxypyridinoline), serum and synovial membrane pro-inflammatory markers [tumour necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-17, vascular endothelial growth factor (VEGF)] and matrix metalloproteinase 1 (MMP1) were measured. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface were also done. RESULTS: Physical parameters, urinary markers, serum and synovial membrane pro-inflammatory makers and MMP1 were increased in arthritic rats and significantly restored after GNP-NKCT1/NKCT1 treatment. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface also indicated the protective effect of GNP-NKCT1 against inflammatory response and cartilage degradation in osteoarthritic rats. INTERPRETATION & CONCLUSIONS: In this study restoration of the arthritic markers and bone degradation by GNP-NKCT1 treatment indicated the anti-osteoarthritic property of GNP-NKCT1. Further studies need to be done to confirm these findings.

Inhibition of complement C3 might rescue vascular hyporeactivity in a conscious hemorrhagic shock rat model.

BACKGROUND: Vascular hyporeactivity in severe hemorrhagic shock could induce refractory hypotension and is an important cause of death. The global acute inflammatory response induced in shock triggers the over-expression of reactive oxygen species, NO, ET1 and TNF-α, which play essential roles in the pathology of vascular hyporeactivity. This leads to a hypothesis that inhibition of the complement system, the mediator of the inflammatory cascade, might be a promising therapeutic exploration for vascular hyporeactivity. METHODS: We use cobra venom factor (CVF) and the soluble form of CR1 (sCR1) which deplete or inhibit complement C3 respectively to examine its role in vascular hyporeactivity in a conscious hemorrhagic shock rat model. RESULTS: We first confirmed the over-activation of C3 during shock and the down-regulation effects of CVF and sCR1 on C3. Then, both CVF and sCR1 could significantly mitigate the over-expression of serum NO, ET-1, TNF-α and reactive oxygen species. Finally, the vascular reactivity of superior mesenteric arteries (SMA) was examined in vitro, which confirmed the massive reduction of vascular reactivity in shock, which was significantly rescued by both CVF and sCR1. CONCLUSIONS: Inhibition of C3 might improve the reactivity of SMA to norepinephrine during hemorrhagic shock possibly through the downregulation of NO, ET1, TNF-α and reactive oxygen radicals.

Therapeutic efficacy and molecular mechanisms of snake (Walterinnesia aegyptia) venom-loaded silica nanoparticles in the treatment of breast cancer- and prostate cancer-bearing experimental mouse models.

The treatment of drug-resistant cancer is a clinical challenge, and thus screening for novel anticancer drugs is critically important. We recently demonstrated a strong enhancement of the antitumor activity of snake (Walterinnesia aegyptia) venom (WEV) in vitro in breast carcinoma, prostate cancer, and multiple myeloma cell lines but not in normal cells when the venom was combined with silica nanoparticles (WEV+NP). In the present study, we investigated the in vivo therapeutic efficacy of WEV+NP in breast cancer- and prostate cancer-bearing experimental mouse models. Xenograft breast and prostate tumor mice models were randomized into 4 groups for each cancer model (10 mice per group) and were treated with vehicle (control), NP, WEV, or WEV+NP daily for 28 days post tumor inoculation. The tumor volumes were monitored throughout the experiment. On Day 28 post tumor inoculation, breast and prostate tumor cells were collected and either directly cultured for flow cytometry analysis or lysed for Western blot and ELISA analysis. Treatment with WEV+NP or WEV alone significantly reduced both breast and prostate tumor volumes compared to treatment with NP or vehicle alone. Compared to treatment with WEV alone, treatment of breast and prostate cancer cells with WEV+NP induced marked elevations in the levels of reactive oxygen species (ROS), hydroperoxides, and nitric oxide; robust reductions in the levels of the chemokines CXCL9, CXCL10, CXCL12, CXCL13, and CXCL16 and decreased surface expression of their cognate chemokine receptors CXCR3, CXCR4, CXCR5, and CXCR6; and subsequent reductions in the chemokine-dependent migration of both breast and prostate cancer cells. Furthermore, we found that WEV+NP strongly inhibited insulin-like growth factor 1 (IGF-1)- and epidermal growth factor (EGF)-mediated proliferation of breast and prostate cancer cells, respectively, and enhanced the induction of apoptosis by increasing the activity of caspase-3,-8, and -9 in both breast and prostate cancer cells. In addition, treatment of breast and prostate cancer cells with WEV+NP or WEV alone revealed that the combination of WEV with NP robustly decreased the phosphorylation of AKT, ERK, and IκBα; decreased the expression of cyclin D1, surviving, and the antiapoptotic Bcl-2 family members Bcl-2, Bcl-XL, and Mcl-1; markedly increased the expression of cyclin B1 and the proapoptotic Bcl-2 family members Bak, Bax, and Bim; altered the mitochondrial membrane potential; and subsequently sensitized tumor cells to growth arrest. Our data reveal the therapeutic potential of the nanoparticle-sustained delivery of snake venom against different cancer cell types.

In vivo evaluation of homeostatic effects of Echis carinatus snake venom in Iran

Background: The venom of the family Viperidae, including the saw-scaled viper, is rich in serine proteinases and metalloproteinases, which affect the nervous system, complementary system, blood coagulation, platelet aggregation and blood pressure. One of the most prominent effects of the snake venom of Echis carinatus (Ec) is its coagulation activity, used for killing prey. Materials and methods: Subfractions F1A and F1B were isolated from Ec crude venom by a combination of gel chromatography (Sephadex G-75) and ion exchange chromatography on a DEAE-Sepharose (DE-52). These subfractions were then intravenously (IV) injected into NIH male mice. Blood samples were taken before and after the administration of these subfractions. Times for prothrombin, partial thromboplastin and fibrinogen were recorded. Results and conclusions: Comparison of the prothrombin time before and after F1A and F1B administrations showed that time for blood coagulation after injection is shorter than that of normal blood coagulation and also reduced coagulation time after Ec crude venom injection. This difference in coagulation time shows the intense coagulation activity of these subfractions that significantly increase the coagulation cascade rate and Causes to quick blood coagulation. The LD50 of the Ec crude venom was also determined to be 11.1 µg/mouse. Different crude venom doses were prepared with physiological serum and injected into four mice. Comparison of the prothrombin times after injection of subfractions F1A and F1B showed that the rate of mouse blood coagulation increases considerably. Comparing the partial thromboplastin times after injecting these subfractions with this normal test time showed that the activity rate of intrinsic blood coagulation system rose sharply in mice. Finally, by comparing the fibrinogen time after subfraction injections and normal test time, we can infer intense activation of coagulation cascade and fibrin production.(AU)

Therapeutic potential of Naja naja atra venom in a rat model of diabetic nephropathy.

OBJECTIVE: To study the protective effects of naja naja atra venom (NNAV) in a rat model of diabetic nephropathy (DN). METHODS: The rat diabetes model was induced by intraperitoneal injection of streptozotocin (STZ). Thirty-two model rats were randomly divided into one DN group (n=8) and three treatment groups (n=8 each) that received NNAV at doses of 30, 90, or 270 µg/(kg·day) via oral gavage, another eight rats as normal controls. After 12 weeks, all rats were sacrificed and the changes in serum and urine biological index levels were determined by colorimetric assay. Microalbumin (mALB), N-acetyl-ß- glucosaminidase (NAG) and cystatin C (CysC) concentrations were measured by ELISA. Renal tissues were sliced for pathological and immunohistochemical observations. RESULTS: Comparied with the DN group, serum glucose was decreased by 31.04%, total cholesterol 21.96%, triglyceride 23.78%, serum creatinine 19.83%, blood urea nitrogen 31.28%, urinary protein excretion 45.42%, mALB 10.42%, NAG 20.65%, CysC 19.57%, whereas albumin increased by 5.55%, high-density lipoprotein-cholesterol 59.09%, creatinine clearance 19.05% in the treatment group by NNAV administration at dose of 90 µg/(kg·day). NNAV also reduced the levels of malondialdehyde in serum (22.56%) and kidney tissue (9.79%), and increased superoxide dismutase concentration in serum (15%) and decreased it in renal tissue (8.85%). In addition, under light microscopy kidney structure was improved and glomerular hypertrophy decreased by 8.29%. As shown by immunohistochemistry, NNAV inhibited transforming growth factor-ß1 by 6.70% and nuclear actor-κB by 5.15%. CONCLUSION: NNAV improves biological indexes in DN, and it may exert renoprotective effects in rats with STZ-induced diabetes.

Black mamba venom peptides target acid-sensing ion channels to abolish pain.

Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

Development, characterization and anti-tumor effect of a sequential sustained-release preparation containing ricin and cobra venom cytotoxin.

Cobra venom cytotoxin (CVC) loaded in poly (lactide-co-glycolide) (PLGA) microspheres was mixed with ricin and encapsulated in a thermosensitive PLGA-PEG-PLGA hydrogel for this study. This sequential sustained-release preparation (SSRP) containing ricin and CVC could avoid burst release effect of CVC from microspheres. In addition, in SSRP, the two biotoxins have different drug release rates and antitumor mechanisms, which can be complementary to each other. Ricin has a faster release rate than CVC. It can combine with the tumor cell membrane and enter the cell, inhibiting protein synthesis within 2 weeks. Whereas CVC releases slowly in 5 weeks directly dissolving the tumor cell membrane and killing the cells which are less-sensitive to ricin. The in vivo experiments showed that intratumoral injection of SSRP could inhibit hepatocellular carcinoma growth significantly, and the tumor growth inhibition rate reached 73.5%. It appears that a new medicine preparation for cancer local treatment should be further studied for clinical applications.

Behavioral and morphological evidence for the involvement of glial cells in the antinociceptive effect of najanalgesin in a rat neuropathic pain model.

Neuropathic pain is a devastating neurological disease that seriously affects patients' quality of life. Despite a high level of incidence, the underlying mechanisms of neuropathic pain are still poorly understood. However, recent evidence supports the prominent role of spinal glial cells in neuropathic pain states. In our laboratory, we observed that najanalgesin, a novel peptide isolated from the venom of Naja naja atra, exerts significant analgesic effects on acute pain in mice and neuropathic pain in rats. The objective of the present study was to determine whether spinal glia are associated with the antinociceptive effect of najanalgesin in an L5 spinal nerve ligation (SNL) rodent model of neuropathic pain. Mechanical allodynia developed after surgery, and hypersensitivity was significantly attenuated by the intrathecal administration of najanalgesin. The inhibitory effect of najanalgesin was significantly (p<0.05) enhanced after pretreatment with fluorocitrate (a glial cell antagonist). In addition, the astrocyte activation was attenuated following najanalgesin treatment in the dorsal horn of neuropathic rats, as assessed by immunohistology and Western blotting. The tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) content of cerebral spinal fluid and cell culture supernatants changed significantly after najanalgesin administration. The results suggest that najanalgesin may exert its anti-allodynic effect by altering astrocyte cell function.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome.

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

[Effects of intratumoral injection of microspheres containing cobra venom cytotoxin on transplanted human hepatoma in nude mice].

OBJECTIVE: To evaluate the safety and efficacy of intratumoral injection of polylactic-co-glycolic acid (PLGA) microspheres containing cobra venom cytotoxin in nude mice with transplanted human hepatoma. METHODS: Cytotoxic activity of cytotoxin from cobra venom was determined by using methyl thiazolyl tetrazolium method in vitro. Microspheres containing cobra venom cytotoxin were prepared with a double emulsion-solvent evaporation method. Forty BALB/c nude mice were inoculated subcutaneously in right flank with hepatoma BEL-7404 cells. Thirty-two mice whose tumor size reached about 1.0 cm in diameter, were randomly assigned into normal saline group, blank microsphers group, cytotoxin group and cytotoxin-PLGA group. Nude mice were intratumorally injected with normal saline, blank microspheres, cytotoxin or cytotoxin-PLGA microspheres respectively. Internal echo characteristics and blood flow of tumors were observed by high-frequency ultrasound every week after treatment. Twenty-six days after treatment, the tumors were removed to calculate the inhibition rate of tumor growth. The tumor, heart, liver and kidney tissues were obtained for histopathological examination. RESULTS: The cytotoxin separated and purified from crude cobra venom caused intense cytotoxic effects to the BEL-7404 cells in vitro. The diameter of PLGA microspheres containing cobra venom cytotoxin was about (34.45+/-9.85)microm. Encapsulation rate was up to (78.13+/-8.92)%, and cumulative amount of cobra venom cytotoxin released from the PLGA microspheres in vitro during 30 days was up to 84.3%. After intratumoral injection, tumor volumes and weights in the cytotoxin-PLGA group were lower than those in the normal saline group, with a tumor growth inhibition rate of 52.36%. Observed under a light microscope, most tumor tissues were necrotic. No obvious morphological change could be seen on the liver, kidney and heart tissues. CONCLUSION: The above findings indicate that intratumoral injection of cytotoxin-PLGA microspheres has strong antitumor effect and can obviously lessen systemic toxicity, which may provide an effective and feasible method for hepatocellular carcinoma treatment.

Differential involvement of M1-type and M4-type muscarinic cholinergic receptors in the dorsomedial striatum in task switching.

Previous experiments have demonstrated that the rat dorsomedial striatum is one brain area that plays a crucial role in learning when conditions require a shift in strategies. Further evidence indicates that muscarinic cholinergic receptors in this brain area support adaptations in behavioral responses. Unknown is whether specific muscarinic receptor subtypes in the dorsomedial striatum contribute to a flexible shift in response patterns. The present experiments investigated whether blockade of M1-type and/or M4-type cholinergic receptors in the dorsomedial striatum underlie place reversal learning. Experiment 1 investigated the effects of the M1-type muscarinic cholinergic antagonist, muscarinic-toxin 7 (MT-7) infused into the dorsomedial striatum in place acquisition and reversal learning. Experiment 2 investigated the effects of the M4-type muscarinic cholinergic antagonist, muscarinic-toxin 3 (MT-3) injected into the dorsomedial striatum in place acquisition and reversal learning. All testing occurred in a modified cross-maze across two consecutive sessions. Bilateral injections of MT-7 into the dorsomedial striatum at 1 or 2 microg, but not 0.05 microg impaired place reversal learning. Analysis of the errors revealed that MT-7 at 1 and 2 microg significantly increased regressive errors, but not perseverative errors. An injection of MT-7 2 microg into the dorsomedial striatum prior to place acquisition did not affect learning. Experiment 2 revealed that dorsomedial striatal injections of MT-3 (0.05, 1 or 2 microg) did not affect place acquisition or reversal learning. The findings suggest that activation of M1-type muscarinic cholinergic receptors in the dorsomedial striatum, but not M4-type muscarinic cholinergic receptors facilitate the flexible shifting of response patterns by maintaining or learning a new choice pattern once selected.

Venom of Indian monocellate cobra and Russell's viper show anticancer activity in experimental models.

Indian monocellate cobra (Naja kaouthia) and Russell's viper (Vipera russelli) are common snakes of the East Indian sub-peninsula. The anticarcinogenic activities of their crude venoms were studied on carcinoma, sarcoma and leukemia models. Sub-lethal doses of venoms showed cytotoxicity on Ehrlich ascites carcinoma (EAC) cells in vivo. The venoms increased lifespan of EAC mice and strengthened the impaired host antioxidant system. Sarcoma formation in mice (3-methylcholanthrene induced) after venom treatment was significantly less (p < 0.005). Histopathological examination of tumors showed tissue necrosis. The venoms displayed potent cytotoxic and apoptogenic effect on human leukemic cells (U937/K562). The venoms reduced cell proliferation rate (p < 0.005) and produced morphological alterations indicative of apoptosis induction. Different degree and nature of anticarcinogenic property of cobra and viper venoms may be attributed to the difference in their constituents.

Complement inhibitors selectively attenuate injury following administration of cobra venom factor to rats.

Systemic activation of complement is a pathophysiological response common to severe disturbances such as hemorrhagic shock, major burn injury and sepsis. Intravenous infusion of cobra venom factor (CVF) has been used as an animal model of acute respiratory distress syndrome (ARDS), and reliably and selectively induces rapid intravascular activation of the complement system, leading to acute organ damage. In the present study, we have used different complement inhibitors to investigate the roles of complement products in CVF-induced responses in the rat. Rats were treated with either a C5a receptor antagonist (C5aRA, AcF-[OP(d-Cha)WR], 1 mg/kg i.v. or 10 mg/kg p.o.), a C3a receptor antagonist (C3aRA, N(2)-[(2,2-diphenylethoxy)acetyl]-l-arginine, 0.1 mg/kg i.v.) or a convertase inhibitor, rosmarinic acid (RMA, 10 mg/kg i.v.), prior to CVF-induced complement challenge. Intravenous CVF resulted in hallmark events evident in the development of ARDS, including systemic neutropenia followed by neutrophil migration to the lung and bronchoalveolar vascular leakage, blood pressure alterations, and an increase in TNFalpha levels in both serum and bronchoalveolar lavage fluid. These hemodynamic changes were differentially inhibited by antagonism of C5a receptors, C3a receptors or by inhibition of the entire complement cascade using RMA. This evidence strongly implicates complement factors in the development of lung injury associated with systemic complement activation and identifies complement inhibition as a potential therapeutic target for acute syndromes such as ARDS and other severe systemic shock states mediated by activation of complement.

Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems.

The mechanisms underlying stromal cell supportive functions are incompletely understood but probably implicate a mixture of cytokines, matrix components and cell adhesion molecules. Skeletal muscle uses recruited macrophages to support post-injury regeneration. We and others have previously shown that macrophages secrete mitogenic factors for myogenic cells. Here, we focused on macrophage-elicited survival signals. We demonstrated that: (1) macrophage influx is temporally correlated with the disappearance of TUNEL-positive apoptotic myogenic cells during post-injury muscle regeneration in mice; (2) direct cell-cell contacts between human macrophages and myogenic cells rescue myogenic cells from apoptosis, as assessed by decreased annexin V labelling and caspase-3 activity, and by increased DIOC-6 staining, Bcl-2 expression and phosphorylation of Akt and ERK1/2 survival pathways; (3) four pro-survival cell-cell adhesion molecular systems detected by DNA macroarray are expressed by macrophages and myogenic cells in vitro and in vivo - VCAM-1-VLA-4, ICAM-1-LFA-1, PECAM-1-PECAM-1 and CX3CL1-CX3CR1; (4) macrophages deliver anti-apoptotic signals through all four adhesion systems, as assessed by functional analyses with blocking antibodies; and (5) macrophages more strongly rescue differentiated myotubes, which must achieve adhesion-induced stabilisation of their structure to survive. Macrophages could secure these cells until they establish final association with the matrix.

Cobra snake venom reduces significantly tissue nucleic acid levels in human breast cancer.

OBJECTIVE: To look into the feasibility of using cobra snake venom in suppressing breast cancer tissue through inhibition of nucleic acid synthesis. METHODS: Samples of breast cancerous tissue, obtained from Atomic Energy Medical Center, LMCH, Jamshoro, incubated with varying concentrations of venom and without venom (as controls) were assayed for macromolecular (RNA and DNA) levels RESULTS: No. change in the control samples while a prominent and significant fall in nucleic acid contents has been noted in venom treated tissues. Also, maximum effect was observed with 25ug/ml dose. CONCLUSION: Snake venom strongly inhibited the formation of nucleic acids in the breast cancerous tissues. It may bring a fall in cell proliferation, thus there is hope that venom could be used as an anti-cancerous drug in the future.