Batroxase promotes the effect of NK cell adoptive therapy on lung cancer by enhancing immune cell infiltration.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin acts in concert with NK cells in terms of anti-tumor effects. CD3+/CD56+ NK cells were isolated and cultured from C57BL/6 mouse spleen. NK cells' viability was tested via Lactate dehydrogenase (LDH) assay. Lewis lung cancer cell (1*107 cell/ml) was used to build animal models. All animals were divided into five groups and treated with Batroxobin and NK cells respectively. HE staining was used to detect the pathological morphology of tumor tissue. The contents of fibrinogen and TNF-α in serum were determined by ELISA. The protein expression levels of MMP2, MMP9, VEGF and CD44 in tumor tissues were detected by Western Blot or immunohistochemistry. Compared with Control group, Tumor growth was not significantly affected in the group treated with Batroxobin or NK cells alone, However, tumor growth was significantly inhibited in the NK cell combined with the Batroxobin group. Serum levels of Fbg and TNF-αin mice treated with Batroxobin combined with NK cells dropped significantly, bringing them closer to normal levels. WB results showed that the expression levels of MMP2/9, VEGF and CD44 in Batroxobin combined with NK cell group also significantly decreased. Batroxobin combined with adoptive immunotherapy with NK cells significantly inhibited the growth of Lewis lung cancer in mice.

A prospective comparison of four methods for preventing pacemaker pocket infections.

This study aims to evaluate four pacemaker pocket cleaning methods for preventing implantation-related infections. This single-center trial prospectively randomized 910 patients undergoing first-time pacemaker implantation or replacement into four pocket cleaning methods: hemocoagulase (group A, n = 228), gentamicin (group B, n = 228), hemocoagulase plus gentamicin (group C, n = 227), and normal saline (group D, n = 227). Before implanting the pacemaker battery, the pockets were cleaned with gauze presoaked in the respective cleaning solutions. Then, these patients were followed up to monitor the occurrence of infections for 1 month after implantation. Twelve implantation-related infections occurred in 910 patients (1.32%): four patients from group A (1.75%), three patients from group B (1.32%), two patients from group C (0.88%), and three patients from group D (1.32%) (P > .05). Furthermore, two patients developed bloodstream infections (0.22%), and both of these patients were associated with pocket infection (one patient was from group A, while the other patient was from group C, respectively). No cases of infective endocarditis occurred. The differences in the number of infections in these study groups were not statistically significant. The application of hemocoagulase, gentamicin, hemocoagulase plus gentamicin, or normal saline on the presoaked gauze before implantation was equally effective in preventing pocket-associated infections.

A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study.

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 106 or 2 × 106 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 µL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.

The efficacy and safety of Batroxobin in combination with anticoagulation on cerebral venous sinus thrombosis.

Cerebral venous sinus thrombosis (CVST) is an uncommon subtype of stroke with highly variable clinical presentation. Although anticoagulation with heparin and/or warfarin remains the standard treatment for CVST, treatment failure is still common. This study aims to evaluate the safety and efficacy of Batroxobin in combination with anticoagulation on CVST control. In this retrospective study, a total of 61 CVST patients were enrolled and divided into Batroxobin (n = 23) and control (n = 38) groups. In addition to the same standard anticoagulation in control, patients in the treatment group received Batroxobin 5 BU intravenous infusion (10 BU for the first time) every other day, for a total of three infusions. A higher recanalization rate was found in Batroxobin group (adjusted OR [95% CI] of 2.5 [1.1-5.0], p = 0.028) compared to the control group, especially in patients with high levels of fibrinogen (adjusted OR [95% CI] of 4.7 [1.4-16.7], p = 0.015). Statistically significant differences between the two groups were seen regarding the levels of thrombin time, fibrinogen and D-dimer at each cut-off time point (all p < 0.01). Compared with baseline, NIHSS scores at discharge showed significant improvement in the Batroxobin group [0(0, 4.25)-5(2, 11), p = 0.036]. No significant difference in mRS scores was found between the two groups at discharge or at 6-month outpatient follow-up (all p > 0.05). Additionally, Batroxobin did not increase the risk of intracranial hemorrhage. We conclude that Batroxobin is a potentially safe and effective adjunct therapeutic agent promoting CVST recanalization especially in patients with high level of fibrinogen.

Batroxobin can improve the efficacy of combination therapy for profound sudden sensorineural hearing loss greater than but not less than 100 dB HL.

OBJECTIVE: To investigate the effects of combination therapy with and without batroxobin, and the frequency of batroxobin use on the prognosis of profound sudden sensorineural hearing loss. METHODS: Hearing recovery in the batroxobin group (231 patients) and non-batroxobin group (56 patients) was compared. The correlation between the number of times batroxobin was used and hearing recovery was analysed. RESULTS: The decrease in hearing threshold and overall improvement rate in the batroxobin group with hearing loss exceeding 100 dB HL was significantly higher than that in the non-batroxobin group. There was no linear correlation between the number of times batroxobin was used and the overall improvement rate. Using batroxobin two to three times achieved a therapeutic effectiveness plateau. CONCLUSION: Batroxobin can improve the efficacy of combination therapy for profound sudden sensorineural hearing loss exceeding 100 dB HL, and using batroxobin two to three times yields the maximum overall improvement rate.

Effects of batroxobin on the antithrombotic system in patients with cerebral venous thrombosis: Clues to mechanisms.

BACKGROUND AND PURPOSE: More evidence supports the benefits of batroxobin combined with anticoagulation in correcting acute cerebral venous thrombosis (CVT). The dynamic fluctuations of peripheral blood platelets, fibrinolysis, and coagulation biomarkers during this therapy were analyzed. METHODS: We investigated batroxobin's effects on the antithrombotic system under two regimens. The pretreatment group included patients on anticoagulants for at least 1 week before starting batroxobin. The simultaneous treatment group began both treatments upon admission. The control group received only anticoagulation. Batroxobin was given on alternate days at doses of 10BU, 5BU, and 5BU, totaling three doses. Anticoagulation was continuous. Baseline data were T0; the next day after each batroxobin dose was T1, T2, and T3. Data from these four time points was analyzed. RESULTS: The time-point paired sample T-test results of the pretreatment group [n = 60; mean age (SD), 43.3(16.5); 38 (63.35%) women] showed that batroxobin significantly inhibited ADP-induced platelet aggregation rate (T1-T0: p = 0.015; T2-T0: p = 0.025; T3-T0: p = 0.013), decreased fibrinogen level (T1-T0: p < 0.001; T2-T0: p < 0.001; T3-T0: p < 0.001), and increased D-dimer (T1-T0:p < 0.001; T2-T0: p < 0.001; T3-T0: p < 0.001), TT (T1-T0:p = 0.046; T2-T0: p = 0.003; T3-T0: p < 0.001), and APTT (T1-T0:p = 0.021; T2-T0: p = 0.012; T3-T0: p = 0.026). Compared to the control group, the simultaneous treatment group showed significantly higher TT (T2: p = 0.002; T3: p = 0.004) and D-dimer (T1: p < 0.001; T2: p < 0.001; T3: p < 0.001) values, while fibrinogen (T2: p < 0.001; T3: p < 0.001) levels were significantly lower. Using batroxobin can alleviate the amplitude of changes in coagulation indicators other than TT caused by anticoagulants. The above conclusions are consistent with the results of repeated measurement data analysis. CONCLUSIONS: Batroxobin can significantly inhibit ADP-induced platelet aggregation rate, increase D-dimer, decrease fibrinogen, and prolong TT and APTT in the presence of anticoagulant agents. Using batroxobin can reduce the amplitude of changes in coagulation indicators caused by anticoagulants. These results reveal the potential mechanism of batroxobin combined with anticoagulation in the safe and effective treatment of CVT.

Anti-inflammatory Effect of Batroxobin Combined With Anticoagulation in Patients With Cerebral Venous Thrombosis.

Inflammation is pivotal in the pathogenesis and development of cerebral venous thrombosis (CVT). Herein, we aimed to assess the anti-inflammatory effects of batroxobin combined with anticoagulation in CVT. Participants were categorized into the batroxobin group (batroxobin combined with anticoagulation) and the control group (anticoagulation only). Regression analysis was employed to explore the association between the number of episodes of batroxobin administration and the fluctuation of inflammatory indicators, as well as the proportion of patients with inflammatory indicators that were reduced after batroxobin use. Twenty-three cases (age: 39.9 ± 13.8 years, female: 39.1%) in the batroxobin group and 36 cases (40.3 ± 9.6 years, 52.8%) in the control group were analyzed. Compared to the control group, batroxobin combined with anticoagulation significantly decreased fibrinogen (P < .001), platelet-lymphocyte ratio (PLR) (P = .016) and systemic immune-inflammation index (SII) (P = .008), and increased the proportion of the patients with lower fibrinogen (P < .001), neutrophil-lymphocyte ratio (NLR) (P = .005), PLR (P = .026), and SII (P = .006). Linear analysis showed that as the number of episodes of batroxobin administration increased, the fibrinogen (P < .001), the PLR (P = .001), and the SII (P = .020) significantly decreased. Logistic regression analysis showed as the number of episodes of batroxobin administration increased, the ratio of the patients with decreased NLR (P = .008) and PLR (P = .015), as well as SII (P = .013), significantly increased. Batroxobin could decrease NLR, PLR, and SII in CVT. The effect was related to the number of episodes of batroxobin administration. Besides reducing fibrinogen and indirect thrombolysis effects, this may be another critical benefit of batroxobin for CVT.

Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin.

BACKGROUND: The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (µm2), the area of the fibrin fibers (µm2), and the width of the fibrin fibers (µm) were determined for the dog clots. The platelet area (µm2), the area of fibrin fibers (µm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. RESULTS: Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. CONCLUSIONS: The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.

[Pharmacodynamics and pharmacokinetics of batroxobin in Beagle dog].

Healthy Beagle dogs were administrated with batroxobin by intravenous infusion at high, medium and low doses. The study of pharmacodynamics and pharmacokinetics was intended to clarify the relevance of them and provided strong evidence for clinical use of batroxobin. The blood samples were collected after injection based on the time schedule and samples were tested by ELISA method to get the concentration of batroxobin. At the same time, changes of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimmer were tested. The results showed that the concentration of D-D increased significantly after administration compared with that of before administration. The main pharmacokinetic parameters were as follows: t1/2 were (2.27 +/- 0.42) h, (10.65 +/- 2.19) h and (11.01 +/- 3.51) h; C(max) were (11.9 +/- 1.72) ng x mL(-1), (154.53 +/- 12.38) ng x mL(-1) and (172.14 +/- 47.33) ng x mL(-1); AUC(last) were (29.38 +/- 3.69) ng xh x mL(-1), (148.43 +/- 72.85) ng x h x mL(-1) and (599.22 +/- 359.61) ng x h x mL(-1). The elimination of batroxobin was found to be in accord with linear kinetics characteristics. The results of pharmacodynamics showed that D-dimmer level increased significantly after the administration of batroxobin, which was similar with the changes of batroxobin plasma concentration. Simultaneously, Fib concentrations in Beagle dog blood decreased significantly after the iv administration of batroxobin, while recovered to base level after 48 hours. PT, TT and APTT significantly became longer after administration, which returned to normal level after 48 hours. Especially, the D-dimmer levels and the batroxobin concentration in plasma after intravenous infusion of the drug were synchronized in Beagle dogs. Changes between PD/PK results had obvious correlation, and the D-dimmer levels in plasma can be one of the important monitoring indicators of batroxobin in thrombolytic medication.

[The topically hemostatic effects of batroxobin on the carotid arteries adventitia removal rabbit].

OBJECTIVE: To observe the topically hemostatic effects of batroxobin (BX) in different concentrations on the carotid arteries adventitia removal rabbit. METHODS: 18 rabbits were removed vascular adventitia by collagenase digestion and mechanical dissection, causing capillary hemorrhage. Then all of them were randomly divided into 6 groups: blank control, negative control group, 0.5, 1, 2 and 4 kU/L (U/ml) BX group. The hemostatic time and bleeding volume were observed to compare the hemostatic effect of each group. Haematoxylin-eosin, Masson staining and immunohistochemistry were performed to assure adventitia removed. RESULTS: It was feasible to remove vascular adventitia with collagenase digestion and mechanical dissection. The hemostatic time and bleeding volume were significantly different (P < 0.05) from 0.5 U/ml BX group [(97 ± 20)s,(0.102 ± 0.013)g/cm(2)] of the negative control group[(143 ± 33)s,(0.130 ± 0.023) g/cm(2)]. With the increase of BX concentration, there was a significant difference (P < 0.05) between 2 U/ml BX group (32 ± 13,0.056 ± 0.015) and 1 U/ml BX group (32 ± 13,0.056 ± 0.015), but there was no statistical significance (P > 0.05) between 2 U/ml BX group and 4 U/ml BX group (28 ± 14,0.053 ± 0.012). Thus, the best topical hemostatic concentration of BX was 2 U/ml. CONCLUSION: The topical hemostatic effect of batroxobin is reliable in small area of blood oozing.

Comparison of the effect of calcium gluconate and batroxobin on the release of transforming growth factor beta 1 in canine platelet concentrates.

BACKGROUND: The clinical use of autologous platelet concentrates (also known as platelet-rich plasma) on the field of regenerative therapy, in the last decade has been the subject of several studies especially in equine medicine and surgery. The objectives of this study was: 1) to describe and compare the cellular population in whole blood, lower fraction (A) and upper fraction (B) of platelet concentrates, 2) to measure and compare the transforming growth factor beta 1 (TGF-ß1) concentration in plasma and both platelet concentrates after be activated with calcium gluconate or batroxobin plus calcium gluconate and, 3) to determine correlations between cell counts in platelet concentrates and concentrations of TGF-ß1. Blood samples were taken from 16 dogs for complete blood count, plasma collection and platelet concentrates preparation. The platelet concentrates (PC) were arbitrarily divided into two fractions, specifically, PC-A (lower fraction) and PC-B (upper fraction). The Platelet concentrates were analyzed by hemogram. After activated with calcium gluconate or batroxobin plus calcium gluconate, TGF-ß1 concentration was determined in supernatants of platelet concentrates and plasma. RESULTS: There were differences statistically significant (P < 0.05) for the platelet count and leukocyte count and TGF-ß1 concentration between whole blood, plasma and both platelet concentrates. A significant correlation was found between the number of platelets in both platelet concentrates and TGF-ß1 concentration. Platelet collection efficiency was 46.34% and 28.16% for PC-A and PC-B, respectively. TGF-ß1 concentration efficiency for PC activated with calcium gluconate was 47.75% and 31.77%, for PC-A and PC-B, respectively. PC activated with batroxobin plus CG showed 46.87% and 32.24% for PC-A and PC-B, respectively. CONCLUSIONS: The methodology used in this study allows the concentration of a number of platelets and TGF-ß1 that might be acceptable for a biological effect for clinical or experimental use as a regenerative therapy in dogs.

Fibrinogen variant BbetaD432A has normal polymerization but does not bind knob "B".

Fibrinogen residue Bbeta432Asp is part of hole "b" that interacts with knob "B," whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed BbetaD432A has normal polymerization, we hypothesized that Bbeta432Asp is not critical for knob "B" binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from BbetaD432A. Surprisingly, the structure (rfD-BbetaD432A+GH) showed the peptide GHRP was not bound to hole "b." We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking. The turbidity and the rate of gamma-gamma dimer formation for BbetaD432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BbetaD432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than "B:b" interactions. We conclude that hole "b" and "B:b" knob-hole binding per se have no influence on fibrin polymerization.

Fibrinogen depleting agent batroxobin has a beneficial effect on experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) was characterized with widespread demyelination and axonal loss of central nervous system (CNS). Fibrinogen (fibrin) deposition was considered as one of the pathogenesis of MS. Therefore, we explored the effects of fibrinogen depleting agent batroxobin in experimental autoimmune encephalomyelitis (EAE) mice model. Our study showed that prevention and suppression with batroxobin significantly ameliorated clinical severity of EAE, reduced inflammatory cells infiltration, and demyelination, and suppressed the activation of astrocytes and macrophages comprising the CD11b(+) population. Batroxobin treatment leads to reduced expression of p-Akt and increased expression of MBP as compared to control. In addition, batroxobin treatment partly reversed the dendric-like formation of macrophages irritated by fibrinogen in vitro. The reduced severity of EAE mice treated with batroxobin suggests that strategy targeting fibrin as a potential therapy for EAE may be beneficial for the treatment of MS patients.

Slounase, a Batroxobin Containing Activated Factor X Effectively Enhances Hemostatic Clot Formation and Reducing Bleeding in Hypocoagulant Conditions in Mice.

Uncontrolled bleeding associated with trauma and surgery is the leading cause of preventable death. Batroxobin, a snake venom-derived thrombin-like serine protease, has been shown to clot fibrinogen by cleaving fibrinopeptide A in a manner distinctly different from thrombin, even in the presence of heparin. The biochemical properties of batroxobin and its effect on coagulation have been well characterized in vitro. However, the efficacy of batroxobin on hemostatic clot formation in vivo is not well studied due to the lack of reliable in vivo hemostasis models. Here, we studied the efficacy of batroxobin and slounase, a batroxobin containing activated factor X, on hemostatic clot composition and bleeding using intravital microcopy laser ablation hemostasis models in micro and macro vessels and liver puncture hemostasis models in normal and heparin-induced hypocoagulant mice. We found that prophylactic treatment in wild-type mice with batroxobin, slounase and activated factor X significantly enhanced platelet-rich fibrin clot formation following vascular injury. In heparin-treated mice, batroxobin treatment resulted in detectable fibrin formation and a modest increase in hemostatic clot size, while activated factor X had no effect. In contrast, slounase treatment significantly enhanced both platelet recruitment and fibrin formation, forming a stable clot and shortening bleeding time and blood loss in wild-type and heparin-treated hypocoagulant mice. Our data demonstrate that, while batroxobin enhances fibrin formation, slounase was able to enhance hemostasis in normal mice and restore hemostasis in hypocoagulant conditions via the enhancement of fibrin formation and platelet activation, indicating that slounase is more effective in controlling hemorrhage.

Neuroprotective effects of Hemocoagulase Agkistrodon on experimental traumatic brain injury.

Traumatic brain injury (TBI) is the major cause of disability and mortality among young people and is associated with neurodegenerative diseases. However, the available clinical options have limited effectiveness. Here, we investigated the neuroprotective effect of Hemocoagulase Agkistrodon (HCA), a thrombin-like enzyme (TLE) isolated and purified from snake venom. Rats subjected to experimental TBI were administered a single dose of HCA or vehicle 10 min after injury. Neurological function was assessed with modified neurological severity score (mNSS). Brain edema were evaluated by measuring brain water content. Levels of hemoglobin and inflammatory cytokines were detected by Enzyme-linked immunosorbent assay (ELISA). In addition, assays including Evans blue extravasation, Western blot analysis and immunofluorescence staining were utilized to determined blood-brain barrier (BBB) integrity. Our results showed that HCA treatment ameliorated neurological deficits (p < 0.01), alleviated brain edema (p < 0.01) and hemorrhage (p < 0.01), decreased the production of the proinflammatory cytokines IL-1ß (p < 0.01), TNF-α (p < 0.01) and IL-6 (p < 0.05), and increased the anti-inflammatory cytokine IL-10 at the contusion site (p < 0.01). Moreover, HCA administration reduced BBB disruption by regulating expression of tight junction proteins, including ZO-1, occludin and claudin-5 (ps < 0.01). Together, our results demonstrate that HCA might have therapeutic efficacy in acute TBI, suggesting a potential clinical application for mitigating the neuropathological damage associated with TBI.

Use of thromboelastography to monitor effects of the hemocoagulase on the blood coagulation in patients after thoracic surgery.

BACKGROUND: Over the past few years, snake venom thrombin-like preparations which possess the advantages of low toxicity, fast onset, and long-lasting efficacy have been commonly used as hemostatic drugs in clinical surgery. However, recently, cases of hypofibrinogenemia and bleeding after using the hemocoagulase have been reported and cannot be ignored. Our study used thromboelastography (TEG) to monitor the hemocoagulase effects of blood coagulation status in patients after thoracic surgery. METHODS: Patients suffering thoracic surgery in the First Affiliated Hospital of Soochow University between June 2018 and June 2019 were selected and grouped based on the results of postoperative TEG. The patients with low coagulation index (CI) detected by the TEG were set as the low CI group, and randomly selected patients with normal CI were set as the control group and matched in gender, age, and disease type with patients in the low CI group. The general condition, disease type, preoperative blood coagulation routine, type of hemocoagulase used, postoperative blood coagulation status, and blood transfusion status of the two groups were separately analyzed. RESULTS: The preoperative fibrinogen (FIB) content in the low CI group was significantly lower than that in the control group (P<0.01). Of the 43 patients, 41 had no bleeding according to indicators like increased drainage. Two had a bleeding tendency, and were thus clinically discontinued from hemocoagulase and supplemented with FIB, fresh frozen plasma, and cryoprecipitate; their drainage volume was significantly reduced, and reexamination of TEG showed normal coagulation status. CONCLUSIONS: For patients with preoperative low FIB or a lower limit of normal value, hemocoagulase should be used with caution; after using this type of thrombin, applying TEG for the timely monitoring of a patient's coagulation status is optimally recommended.

[Neuroprotective effect of batroxobin on experimental intracerebral hemorrhage in rats].

This study is to investigate if batroxobin has the protective effect against nerve injury caused by cerebral hemorrhage in rats and its possible mechanism. Animals were divided into sham group, model group, batroxobin 4, 8, and 16 BU x kg(-1) groups and nimodipine positive control group. On the brain stereotaxic apparatus, the rat intracerebral hemorrhage model was established by injecting collagenase with microinjector into the brain caudate nucleus which was located according to the brain stereotaxic atlas. Neuroethology of the rats was estimated. The brain tissue pathomorphology was observed with electron microscope. The water content of the brain tissue was quantitated with wet/dry weight measurement. SOD and MDA were determined according to the kit procedure, and free Ca2+ concentration in neurocyte was measured by fluorospectrophotometer. As shown in results, batroxobin could improve neuroethology scale of the rats, relieve histiocyte edema and bleeding degree. The water content of the brain tissue, MDA and free Ca2+ concentration were reduced and SOD activity was raised in batroxobin treatment groups. Therefore, it is possible that batroxobin has some protective effect against nerve injury caused by cerebral hemorrhage in rats, and its mechanism maybe relate to lessening brain edema, reducing MDA content, raising SOD activity, and inhibiting calcium overload.

[Effects of Batroxobin on distal anastomotic intimal hyperplasia after expanded polytetrafluoroethylene bypass grafting in dog common carotid artery].

OBJECTIVE: To study the morphological behavior, the expression of MCP-1 and PDGF-B of distal anastomotic neointima after expanded polytetrafluoroethylene (ePTFE) bypass grafting. And to identify the effects of Batroxobin (BX) on distal anastomotic intimal hyperplasia. METHODS: 12 adult mongrel dogs underwent end to side bypass grafting in left common carotid artery using ePTFE vascular graft (6mm in diameter, 5cm in length) were divided randomly into two groups. All 12 bypass grafts were removed 28 days after grafting and distal anastomotic parts were obtained. Using HE, Masson, IHC, RT-PCR and western blot, we analyzed the thickness, relative content of ECM, SMC PCNA index, expression of MCP-1 and PDGF-B in distal anastomotic neointima. And we compared the differences between the two groups. RESULTS: 28 day after grafting, the neointima was formed by SMC and ECM and was covered by epithelial cells. After using BX, the thickness of neointima was reduced [( 381.3 +/- 144.7) microm versus (213.8 +/- 29.0) microm, P < 0.05] and the relative content of ECM were significantly decreased (P = 0.006). The PCNA index (P = 0.109) and PDGF-B level (P = 0.055 by RT-PCR and P = 0.337 by WB) between the two groups had no significant difference. MCP-1 level was markedly decreased (P = 0.025 by RT-PCR and P = 0.016 by WB). CONCLUSIONS: Using BX after ePTFE grafting could effectively reduce the relative content of ECM, reduce the expression of MCP-1 and decrease the thickness of neointima in the early time.

Effects of batroxobin treatment on the survival of random skin flaps in rats.

OBJECTIVE: Batroxobin is a medicinal preparation extracted from the venom of the Fer-de-Lance snake, and is used to lower blood viscosity, promote blood fibrinogen decomposition, and inhibit thrombosis. This research is to investigate whether batroxobin can improve the survival of random skin flaps in a rat model. MATERIALS AND METHODS: Dorsal McFarlane flaps were harvested from 36 rats divided into two groups. Experimental group: Batroxobin was administered via the tail vein once daily. CONTROL GROUP: The same amount of normal saline was injected instead. On day 2, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured. On day 7, tissue slices were stained with haematoxylin and eosin. Expression of vascular endothelial growth factor (VEGF) was immunohistochemically evaluated. Microcirculatory flow was measured by laser Doppler flowmetry. Flap angiography, using the lead oxide-gelatin injection technique, was performed with the aid of a soft X-ray machine. RESULTS: The batroxobin group exhibited a greater mean flap survival area, a better microcirculatory flow, and higher-level expression of SOD and VEGF compared with the control group. However, the MDA level was significantly reduced. CONCLUSION: Batroxobin effectively improved the survival of random skin flaps.

Immune complex-mediated glomerulonephritis is ameliorated by thrombin-activatable fibrinolysis inhibitor deficiency.

The activity of plasmin plays a critical role in the development of chronic glomerulonephritis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a potent inhibitor of plasmin generation. We hypothesized that TAFI is involved in the pathogenesis of glomerulonephritis because it inhibits plasmin generation. To demonstrate this hypothesis, we compared the development of immune complex-mediated glomerulonephritis in wild-type and TAFI-deficient mice. After six weeks of treatment with horse spleen apoferritin and lipoplysaccharide to induce glomerulonephritis, mice deficient in TAFI had significantly better renal function as shown by lower concentrations of albumin in urine and blood urea nitrogen compared to wild-type mice. In addition, the activity of plasmin and matrix metalloproteinases was significantly increased, and mesangial matrix expansion and the deposition of collagen and fibrin in kidney tissues were significantly decreased in TAFI-knockout mice as compared to their wild-type counterparts. Depletion of fibrinogen by batroxobin (Defibrase) treatment led to equalization of the renal function and the amount of collagen deposition in the kidneys of TAFI-knockout and wild-type mice with immune complex-mediated glomerulonephritis. Together these observations suggest that TAFI-mediated inhibition of plasmin generation plays a role in the pathogenesis of glomerulonephritis, and that it may constitute a novel molecular target for the therapy of this disease.