Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.
Animals ; Apoptosis/drug effects* ; Calpain/antagonists & inhibitors* ; Cyclic GMP/biosynthesis* ; Diabetes Complications/drug therapy* ; Diabetes Mellitus, Experimental/complications* ; Dipeptides/therapeutic use* ; Endothelium/metabolism* ; Enzyme Inhibitors/therapeutic use* ; Erectile Dysfunction/etiology* ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth/metabolism* ; Nitric Oxide Synthase Type III/biosynthesis* ; Penis/enzymology* ; Up-Regulation

BACKGROUNDIn addition to neurons, all components of the neurovascular unit (NVU), such as glial, endothelial, and basal membranes, are destroyed during traumatic brain injury (TBI). Previous studies have shown that excessive stimulation of calpain is crucial for cerebral injury after traumatic insult. The objective of this study was to investigate whether calpain activation participated in NVU disruption and edema formation in a mouse model of controlled cortical impact (CCI).

METHODSOne hundred and eight mice were divided into three groups: the sham group, the control group, and the MDL28170 group. MDL28170 (20 mg/kg), an efficient calpain inhibitor, was administered intraperitoneally at 5 min, 3 h, and 6 h after experimental CCI. We then measured neurobehavioral deficits, calpain activity, inflammatory mediator levels, blood-brain barrier (BBB) disruption, and NVU deficits using electron microscopy and histopathological analysis at 6 h and 24 h after CCI.

RESULTSThe MDL28170 treatment significantly reduced the extent of both cerebral contusion (MDL28170 vs. vehicle group, 16.90 ± 1.01 mm΃ and 17.20 ± 1.17 mm΃ vs. 9.30 ± 1.05 mm΃ and 9.90 ± 1.17 mm΃, both P < 0.001) and edema (MDL28170 vs. vehicle group, 80.76 ± 1.25% and 82.00 ± 1.84% vs. 82.55 ± 1.32% and 83.64 ± 1.25%, both P < 0.05), improved neurological scores (MDL28170 vs. vehicle group, 7.50 ± 0.45 and 6.33 ± 0.38 vs. 12.33 ± 0.48 and 11.67 ± 0.48, both P < 0.001), and attenuated NVU damage resulting (including tight junction (TJ), basement membrane, BBB, and neuron) from CCI at 6 h and 24 h. Moreover, MDL28170 markedly downregulated nuclear factor-κB-related inflammation (tumor necrosis factor-α [TNF-α]: MDL28170 vs. vehicle group, 1.15 ± 0.07 and 1.62 ± 0.08 vs. 1.59 ± 0.10 and 2.18 ± 0.10, both P < 0.001; inducible nitric oxide synthase: MDL28170 vs. vehicle group, 4.51 ± 0.23 vs. 6.23 ± 0.12, P < 0.001 at 24 h; intracellular adhesion molecule-1: MDL28170 vs. vehicle group, 1.45 ± 0.13 vs. 1.70 ± 0.12, P < 0.01 at 24 h) and lessened both myeloperoxidase activity (MDL28170 vs. vehicle group, 0.016 ± 0.001 and 0.016 ± 0.001 vs. 0.024 ± 0.001 and 0.023 ± 0.001, P < 0.001 and 0.01, respectively) and matrix metalloproteinase-9 (MMP-9) levels (MDL28170 vs. vehicle group, 0.87 ± 0.13 and 1.10 ± 0.10 vs. 1.17 ± 0.13 and 1.25 ± 0.12, P < 0.001 and 0.05, respectively) at 6 h and 24 h after CCI.

CONCLUSIONSThese findings demonstrate that MDL28170 can protect the structure of the NVU by inhibiting the inflammatory cascade, reducing the expression of MMP-9, and supporting the integrity of TJ during acute TBI.

Animals ; Brain Injuries, Traumatic ; drug therapy ; metabolism ; Calpain ; antagonists & inhibitors ; metabolism ; Dipeptides ; therapeutic use ; Disease Models, Animal ; Glycoproteins ; therapeutic use ; Inflammation ; drug therapy ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; metabolism ; Peroxidase ; antagonists & inhibitors ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDCalpain, a calcium-dependent cysteine protease, has been demonstrated to regulate osteoclastogenesis, which is considered one of the major reasons for cancer-induced bone pain (CIBP). In the present study, calpain inhibitor was applied in a rat CIBP model to determine whether it could reduce CIBP through regulation of osteoclastogenesis activity.

METHODSA rat CIBP model was established with intratibial injection of Walker 256 cells. Then, the efficacy of intraperitoneal administered calpain inhibitor III (MDL28170, 1 mg/kg) on mechanical withdrawal threshold (MWT) of bilateral hind paws was examined on postoperative days (PODs) 2, 5, 8, 11, and 14. On POD 14, the calpain inhibitor's effect on tumor bone tartrate-resistant acid phosphatase (TRAP) stain and radiology was also carefully investigated.

RESULTSPain behavioral tests in rats showed that the calpain inhibitor effectively attenuated MWTs of both the surgical side and contralateral side hind paws on POD 5, 8, and 11 (P < 0.05). TRAP-positive cell count of the surgical side bone was significantly decreased in the calpain inhibitor group compared with the vehicle group (P < 0.05). However, bone resorption and destruction measured by radiographs showed no difference between the two groups.

CONCLUSIONSCalpain inhibitor can effectively reduce CIBP of both the surgical side and nonsurgical side after tumor injection in a rat CIBP model. It may be due to the inhibition of receptor activator of nuclear factor-kappa B ligand-induced osteoclastogenesis. Whether a calpain inhibitor could be a novel therapeutic target to treat CIBP needs further investigation.

Animals ; Bone Neoplasms ; drug therapy ; physiopathology ; Dipeptides ; therapeutic use ; Disease Models, Animal ; Gene Expression Regulation, Neoplastic ; drug effects ; Glycoproteins ; therapeutic use ; Male ; Osteogenesis ; drug effects ; Pain ; drug therapy ; etiology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDBrain dysfunction is a frequent complication of sepsis, usually defined as sepsis-associated encephalopathy (SAE). Although the Notch signaling pathway has been proven to be involved in both ischemia and neuronal proliferation, its role in SAE is still unknown. Here, the effect of the Notch signaling pathway involved γ-secretase inhibitor DAPT on SAE in septic rats was investigated in a cecal ligation and puncture (CLP) model.

METHODSFifty-nine Sprague-Dawley rats were randomly divided into four groups, with the septic group receiving the CLP operation. Twenty-four hours after CLP or sham treatment, rats were sacrificed and their hippocampus was harvested for Western blot analysis. TNF-α expression was determined using an enzyme-linked immunosorbent assay (ELISA) kit. Neuronal apoptosis was assessed by TUNEL staining, and neuronal cell death was detected by H&E staining. Finally, a novel object recognition experiment was used to evaluate memory impairment.

RESULTSOur data showed that sepsis can increase the expression of hippocampal Notch receptor intracellular domain (NICD) and poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1), as well as the inflammatory response, neuronal apoptosis, neuronal death, and memory dysfunction in rats. The γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT) can significantly decrease the level of NICD and PARP-1, reduce hippocampal neuronal apoptosis and death, attenuate TNF-α release and rescue cognitive impairment caused by CLP.

CONCLUSIONThe neuroprotective effect of DAPT on neuronal death and memory impairment in septic rats, which could be a new therapeutic approach for treating SAE in the future.

Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Animals ; Apoptosis ; drug effects ; Dipeptides ; therapeutic use ; Hippocampus ; drug effects ; metabolism ; Male ; Neurons ; cytology ; drug effects ; Neuroprotective Agents ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Notch ; metabolism ; Sepsis ; complications ; Sepsis-Associated Encephalopathy ; drug therapy ; enzymology ; Signal Transduction ; drug effects

OBJECTIVETo explore the clinical effect of L-ornithine L-aspartate (LOLA) granules in treating chronic liver disease in patients with high-level serum gamma-glutamyltransferase (G-GT) using a 24-week treatment course.

METHODSTwo-hundred patients with chronic liver disease and above normal G-GT were given a 12-week course of LOLA granules (9 g/d) and then classified into the following three groups according to the change in serum Gamma-GT:group I:patients with Gamma-GT level returned to normal;group II:patients with serum Gamma-GT level that was reduced during the treatment; group III:patients with serum Gamma-GT level that did not decrease or that increased to a higher level than at start of treatment.After the 12-week treatment course, the patients in group I were divided into three subgroups for receipt of a control drug (compound glycyrrhizin, 50mg/d) or an additional 12-week course of Gamma-GT at a reduced dose (LOLA granules 3 g/d) or at the original dose; groups II and III were maintained on the initial dose for an additional 12 weeks.The groups were reassessed at the end of the second 12-week course (at the end of week 24 of the study's observation period).Count data were compared using the x2 test and measurement data were compared using the t-test.

RESULTSIn group I, the serum Gamma-GT level was 90.9% at the end of the first 12-week course and dropped to a mean level of 52.2% for both of the subgroups that received the reduced and original dose after the additional 12 weeks of LOLA granules treatment; the difference from week 12 to week 24 was significant (x2=8.213, P less than 0.05).The 24-week change in serum Gamma-GT levels for the group I reduced and original dose subgroups vs.the control subgroup were also significantly different from those seen in groups II and III (P less than 0.05).The percentage of patients in group I who achieved normal level serum Gamma-GT after 24 weeks of treatment (78.6%) was significantly higher than that for the control group (vs.55.0%, x2=11.452, P less than 0.05).When the patients in group 1 who had received the 12 additional weeks of LOLA granules treatment were measured again at two weeks after the treatments had been discontinued (end of week 26), the percentage of patients with normal serum Gamma-GT level was 92.7%, with only three cases showing obviously abnormal levels; in contrast, the group I patients in the control group of the second 12-week study period had on 66.7% of patients with normal-level serum Gamma-GT.The difference in change between the treated groups (both reduced and original dose) and the control group was significant (x2=14.964, P less than 0.05).

CONCLUSIONPatients whose serumGamma-GT levels returned to normal after receipt of LOLA granules for 12 weeks benefitted from an additional 12 weeks of consolidation treatment, and those given the treatment at the original dose benefitted most.Compared with the compound glycyrrhizin, LOLA granules provided a better maintenance of resolved Gamma-GT level.Therefore, the effect of LOLA appears to be reliable and stable as well as safe for clinical use.

Chronic Disease ; Dipeptides ; therapeutic use ; Humans ; Liver Diseases ; drug therapy ; Liver Function Tests ; gamma-Glutamyltransferase ; blood

OBJECTIVETo investigate the therapeutic efficacy and safety of aspartate-ornithine granules in patients with nonalcoholic steatohepatitis (NASH).

METHODSSeventy-two patients with NASH were included in this multiple-dose parallel controlled clinical trial and received a 12-week course of aspartate-ornithine granule treatment at either high-dose (6 g bid po; n = 38) or low-dose (3 g bid po; n = 34). Clinical efficacy was assessed by monitoring data from urinalysis, serologic tests (alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and triglyceride (TG)), and abdominal computed tomography (CT) scan. Safety was assessed by occurrence of adverse events (fatigue, anorexia, abdominal distension, nausea, and vomiting). Statistical analyses were conducted to determine the significance of differences between parameters before (baseline) and after treatment.

RESULTSAfter 12 weeks of treatment, the liver and spleen CT ratios in both the high-dose group (0.89 +/- 0.19) and the low-dose group (0.80 +/- 0.15) were significantly higher than at baseline (S = 329, P less than 0.0001 and S = 246, P less than 0.0001); the overall improvement was more robust in the high-dose group (52.63%) than in the low-dose group (38.23%) (Z = -2.1042, P less than 0.05). After 6 and 12 weeks of treatment, the serum ALT levels in both the high-dose group and the low-dose group were significantly lower than at baseline (6 weeks: S = 324.5, P less than 0.0001 and S = 223, P less than 0.0001; 12 weeks: S = 370.5, P less than 0.0001 and S = 297.5, P less than 0.0001); the overall improvement was more robust in the high-dose group (79.0%) than in the low-dose group (53.0%) (Z = -2.0533, P less than 0.05). Similar trends were seen for the serum levels of AST and GGT after 6 and 12 weeks of treatment (all P less than 0.01) and serum levels of TG after 12 weeks of treatment. The rate of adverse reactions was low and similar between the two groups (high-dose: 4.8% and low-dose: 4.4%; all gastrointestinal).

CONCLUSIONAspartate-ornithine granule therapy was an effective and safe treatment of nonalcoholic steatohepatitis, with the higher dose of 6 g bid po providing more robust clinical benefit without affecting the safety profile.

Adult ; Alanine Transaminase ; blood ; Aspartate Aminotransferases ; blood ; Dipeptides ; administration & dosage ; therapeutic use ; Dose-Response Relationship, Drug ; Female ; Humans ; Male ; Middle Aged ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Treatment Outcome ; Triglycerides ; blood ; gamma-Glutamyltransferase ; blood

OBJECTIVETo investigate the potential effects on cognitive function, prognosis, and neuropeptide levels of patients in response to combination therapy with ornithine aspartate plus naloxone for hepatic encephalopathy.

METHODSEighty-four consecutive patients diagnosed with hepatic encephalopathy were randomly divided into two equal groups. The control group (n = 42) received traditional medical treatment, and the research group (n = 42) received the traditional medical treatment as well as the combination therapy with ornithine aspartate plus naloxone. The supplemental treatment was comprised of daily intravenous injection of 10-15 g ornithine aspartate in 250 ml of 5% glucose plus intravenous drip of 3 mg naloxone in 100 ml of 5% glucose, and was given in 7-day cycles for one or two cycles. The cognitive function of patients was assessed by Hasegawa Intelligence Scale (HDS) and Mini-Mental State Examination (MMSE) questionnaires. The effective rate and time duration from coma to consciousness were recorded. Changes in blood ammonia level, markers of liver function, and neuropeptide levels were measured by standard biochemical assays. Intergroup differences were assessed by the Chi-squared test.

RESULTSThe HDS and MMSE scores of the research group were significantly higher than those of the control group after therapy. The effective rate, time duration from coma to consciousness, blood ammonia, the liver function markers alanine aminotransferase, gamma-glutamyl-transpeptidase and total bilirubin, and the neuropeptides arginine vasopressin and beta-endorphin were remarkably improved after treatment in the research group, as compared with that in the control group.

CONCLUSIONSupplementing the traditional treatment for hepatic encephalopathy with ornithine aspartate plus naloxone combination therapy provides better therapeutic outcome than traditional treatment alone.

Adult ; Dipeptides ; therapeutic use ; Female ; Hepatic Encephalopathy ; drug therapy ; metabolism ; psychology ; Humans ; Male ; Middle Aged ; Naloxone ; therapeutic use ; Neuropeptides ; metabolism ; Prognosis

Dipeptides ; administration & dosage ; therapeutic use ; End Stage Liver Disease ; drug therapy ; virology ; Female ; Hepatitis B virus ; Humans ; Male

OBJECTIVETo observe the effects of Tangshen Recipe (TR) on the homocysteine (Hcy) metabolism of patients with diabetic nephropathy.

METHODS64 patients with diabetic nephropathy were randomly assigned to two groups, 32 in each. Those in the Western medicine treatment group (Group A) received insulin and orally took anti-diabetic drugs, while those in the TR group (Group B) received insulin and orally took TR (consisting of astragalus, raw rehmannia root, sanchi root, euonymus branchlet, rhubarb, bitter orange, and dogwood fruit, etc. 4 g/package). Six months was taken as one therapeutic course. Another 48 healthy volunteers were recruited as the control group. Eight intermediate metabolites of the homocysteine metabolism in plasma were quantitated before treatment, three months and six months after treatment. The in vivo changes of each metabolite after treatment were analyzed.

RESULTSCompared with the healthy control group, contents of cysteine (Cys), Hcy, s-adenosylmethionine (SAM), and SAH significantly increased in Group A and B before treatment. Contents of methionine (Met), glutathione (GSH), and Cys-gly decreased significantly, showing statistical difference (P<0.05). Patients' in vivo contents of Cys, Hcy, SAM, and SAH significantly decreased, while contents of Met, GSH, and Cys-gly significantly increased after three and six months of treatment when compared with before treatment in the same group (all P<0.05). No statistical difference existed in contents of SAH, SAM, and GSH of Group A and B after six months of treatment when compared with the healthy control group (P>0.05). No adverse reaction occurred in Group A and B.

CONCLUSIONSTreatment of insulin and TR showed similar favorable effect to Western medicine in treatment of diabetic nephropathy. It could improve in vivo hypomethylation and oxidative stress.

Adult ; Aged ; Case-Control Studies ; Diabetic Nephropathies ; drug therapy ; metabolism ; Dipeptides ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Homocysteine ; blood ; metabolism ; Humans ; Insulin ; therapeutic use ; Male ; Middle Aged ; Phytotherapy

BACKGROUNDCathespin-B (cath-B) is an important proteolytic enzyme involved in the disease course of invasion in many types of cancer. Cath-B expression in subcutaneous heteroplastic pancreatic carcinoma in nude mice has not been studied. We investigated the role of cath-B in a model of heteroplastic pancreatic carcinoma in BALB/c nude mice.

METHODSThirty-two six-week-old female BALB/c nude mice were equally divided into four groups. PANC-1 cells were inoculated subcutaneously in the left axillary region. Besides volume, weight of subcutaneous tumor, and change in body weight, cath-B expression in each group was measured by immunohistochemical staining, PCR and Western blotting. Its relationship to microvessel density (MVD), CD44v6, and placenta growth factor (PLGF) was also examined. CA-074Me, a specific inhibitor of cath-B, was injected intraperitoneally (i.p.) at different stages of tumor growth in group B and C. Gemcitabine (GEM), was also injected (i.p.) in group D to compare anti-tumor efficacy with CA-074Me.

RESULTSExpression of cath-B at different levels was related to tumor growth, MVD, and PLGF expression. In group A (control group), cath-B expression was enhanced more than that seen in other groups. CA-074Me clearly inhibited cath-B expression and tumor growth in group B. There was no difference between group C and D with respect to anti-tumor effect.

CONCLUSIONSCath-B correlates with the growth and angiogenesis of tumors, but not with the adhesion induced by CD44v6. CA-074Me clearly inhibited cath-B expression and demonstrated an anti-neoplastic and anti-angiogenesis effect.

Animals ; Antineoplastic Agents ; therapeutic use ; Blotting, Western ; Body Weight ; Cathepsin B ; antagonists & inhibitors ; genetics ; metabolism ; physiology ; Cell Line, Tumor ; Dipeptides ; therapeutic use ; Female ; Humans ; In Vitro Techniques ; Mice ; Mice, Nude ; Pancreatic Neoplasms ; drug therapy ; metabolism ; Placenta Growth Factor ; Pregnancy Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous