The CACNA1A Mutant Disrupts Lysosome Calcium Homeostasis in Cerebellar Neurons and the Resulting Endo-Lysosomal Fusion Defect Can be Improved by Calcium Modulation.

Mutations in P/Q type voltage gated calcium channel (VGCC) lead severe human neurological diseases such as episodic ataxia 2, familial hemiplegic migraine 1, absence epilepsy, progressive ataxia and spinocerebellar ataxia 6. The pathogenesis of these diseases remains unclear. Mice with spontaneous mutation in the Cacna1a gene encoding the pore-forming subunit of P/Q type VGCC also exhibit ataxia, epilepsy and neurodegeneration. Based on the previous work showing that the P/Q type VGCC in neurons regulates lysosomal fusion through its calcium channel activity on lysosomes, we utilized CACNA1A mutant mice to further investigate the mechanism by which P/Q-type VGCCs regulate lysosomal function and neuronal homeostasis. We found CACNA1A mutant neurons have reduced lysosomal calcium storage without changing the resting calcium concentration in cytoplasm and the acidification of lysosomes. Immunohistochemistry and transmission electron microscopy reveal axonal degeneration due to lysosome dysfunction in the CACNA1A mutant cerebella. The calcium modulating drug thapsigargin, by depleting the ER calcium store, which locally increases the calcium concentration can alleviate the defective lysosomal fusion in mutant neurons. We propose a model that in cerebellar neurons, P/Q-type VGCC maintains the integrity of the nervous system by regulating lysosomal calcium homeostasis to affect lysosomal fusion, which in turn regulates multiple important cellular processes such as autophagy and endocytosis. This study helps us to better understand the pathogenesis of P/Q-type VGCC related neurodegenerative diseases and provides a feasible direction for future pharmacological treatment.

Liver-targeted delivery of asiatic acid nanostructured lipid carrier for the treatment of liver fibrosis.

Liver fibrosis is a major global health concern. Management of chronic liver disease is severely restricted in clinics due to ineffective treatment approaches. However, a lack of targeted therapy may aggravate this condition. Asiatic acid (AA), a pentacyclic triterpenoid acid, can effectively protect the liver from hepatic disorders. However, the pharmaceutical application of AA is limited by low oral bioavailability and poor targeting efficiency. This study synthesized a novel liver-targeting material from PEG-SA, chemically linked to ursodeoxycholic acid (UA), and utilized it to modify AA nanostructured lipid carriers (UP-AA-NLC) with enhanced targeting and improved efficacy. The formulation of UP-AA-NLC was optimized via the Box-Behnken Experimental Design (BBD) and characterized by size, zeta potential, TEM, DSC, and XRD. Furthermore, in vitro antifibrotic activity and proliferation of AA and NLCs were assessed in LX-2 cells. The addition of UP-AA-NLC significantly stimulated the TGF-beta1-induced expression of α-SMA, FN1, and Col I α1. In vivo near-infrared fluorescence imaging and distribution trials in rats demonstrated that UP-AA-NLC could significantly improve oral absorption and liver-targeting efficiency. Oral UP-AA-NLC greatly alleviated carbon tetrachloride-induced liver injury and fibrosis in rats in a dosage-dependent manner, as reflected by serum biochemical parameters (AST, ALT, and ALB), histopathological features (H&E and Masson staining), and antioxidant activity parameters (SOD and MDA). Also, treatment with UP-AA-NLC lowered liver hydroxyproline levels, demonstrating a reduction of collagen accumulation in the fibrotic liver. Collectively, optimized UP-AA-NLC has potential application prospects in liver-targeted therapy and holds great promise as a drug delivery system for treating liver diseases.

pH-sensitive chitosan-deoxycholic acid/alginate nanoparticles for oral insulin delivery.

Oral absorption of peptides/proteins is usually compromised by various gastrointestinal tract barriers. To improve delivery efficiency, chitosan-conjugated deoxycholic acid (CS-DCA) coupled with sodium alginate (ALG) was prepared to load insulin into pH-sensitive nanoparticles. The insulin-loaded chitosan-deoxycholic acid/alginate nanoparticles (CDA NPs) were characterized by size (143.3 ± 10.8 nm), zeta potential (19.5 ± 1.6 mV), entrapment efficiency (61.14 ± 1.67%), and insulin drug loading (3.36 ± 0.09%). The CDA NPs exhibited pH-triggered release characteristics in vitro and protected the wrapped insulin from gastric degradation. Stability of the CDA NPs in enzyme-containing simulated gastrointestinal fluids suggested that the NPs could partially protect the wrapped insulin from enzymatic degradation. Additionally, CS-DCA-modified NPs promoted the permeability of Caco-2 cells and enhanced intracellular absorption of FITC-labeled insulin by 9.4 and 1.2-folds, when compared to insulin solution and unmodified NPs, respectively. The positively charged NPs increased intestinal villi adhesion and enhanced insulin absorption in the intestines of diabetic rat models. Furthermore, the hypoglycemic test showed that CDA NPs prolonged insulin release in vivo and exerted a remarkable hypoglycemic effect on diabetic rats with an oral bioavailability of 15%. In conclusion, CDA NPs is a potential oral insulin delivery system.

Comparative pharmacokinetics of tedizolid in rat plasma and cerebrospinal fluid.

To investigate the possibility of tedizolid phosphate's application in the treatment of intracranial infection, a preclinical comparative pharmacokinetic study was designed. Based on the assumption that the classic efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) may participate in the transportation of TDZ, two groups of rats were intravenously administered 6 mg/kg tedizolid phosphate alone or 6 mg/kg tedizolid phosphate combined with 1 mg/kg elacridar which was an inhibitor of P-gp and BCRP. Plasma and cerebrospinal fluid samples were collected according to a pharmacokinetic schedule. All the plasma and cerebrospinal fluid samples were assessed with a validated LC-MS/MS method. The penetration ratio of tedizolid from the blood to cerebrospinal fluid was calculated, and a comparison of the penetration ratios between the two groups was made. The mean Cmax of tedizolid in the CSF in the tedizolid phosphate group and the tedizolid phosphate combined with elacridar group was 154 ng/mL and 300 ng/mL, respectively, and the mean penetration ratio of tedizolid in the tedizolid phosphate group and the tedizolid phosphate combined with elacridar group was 2.16% and 3.53%, respectively. The relatively high Cmax in the CSF proved the possibility of tedizolid phosphate's application in the treatment of intracranial infection, and the higher penetration ratios, Cmax, csf and AUCcsf of the rats in co-administered elacridar group than those in the single-administration group indicated that the transporters P-gp and BCRP might be involved in the transportation of tedizolid.

Stereoselectivity evaluation of chiral chitosan microspheres delivery system containing rac-KET in vitro and in vivo.

The influence of chiral excipient D-chitosan (CS) on the stereoselective release of racemic ketoprofen (rac-KET) microspheres has been investigated in comparison to those microspheres containing individual enantiomers in vitro and in vivo. Stereoselectivity was observed in vitro release test, with R-KET release slightly higher than that of S-KET, especially in 3% rac-KET loading microspheres. Stereoselectivity is dependent on the content of chiral excipient and pH of release medium. A molecular docking study between CS and KET enantiomers further revealed that S-KET has a stronger interaction with CS compared to R-KET. Moreover, the plasma concentration of KET enantiomers in rats shows substantial differences, as the plasma levels of S-KET were higher than those of R-KET. Plasma levels of enantiomers from the R-KET microspheres had similar stereoselectivity as rac-KET microspheres. The S/R ratio of rac-KET microspheres was significantly lower than that of rac-KET suspension (regular-release formulation) (p<.05), and the differences is 3-5 fold. Besides, rates of R-KET converted to S-KET exhibited differences between rac-KET microspheres and suspension. Similar results were also found between R-KET microspheres and suspension. All investigations suggest that the chitosan interacting preferentially with S-KET to R-KET significantly affect the stereoselective pharmacokinetics of rac-KET from chitosan microspheres in rats.

Protective effect of Lycium barbarum polysaccharides against cadmium-induced testicular toxicity in male mice.

The present study was performed to determine whether Lycium barbarum polysaccharides (LBPs) would protect mice against cadmium (Cd)-induced testicular toxicity. Seventy-two male mice were randomly divided into six groups with twelve mice per group. Four groups were administered orally with cadmium chloride (5.0 mg per kg body weight) for 35 days and treated in combination with LBPs (0, 10.0, 33.3 or 100 mg kg-1) from one week before exposure to Cd until the end of the experiment. The other two groups were administered orally with vehicle or LBP (100 mg kg-1) only. Pretreatment with LBP ameliorated the Cd-induced reduction in the body weights, sperm motility as well as the level of testosterone in serum. Moreover, Cd-induced increase in the abnormal sperms was reduced and effects of Cd on the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were reversed. Histopathological examination further confirmed that the LBPs effectively attenuated Cd-induced degeneration of seminiferous tubules. Thus, LBPs attenuated Cd-induced testicular injury by improving the activity of antioxidant enzymatic activity and lowering the oxidative stress, so it could be a potential auxiliary therapeutic agent for Cd-induced testicular toxicity.

Delayed administration of the GLP-1 receptor agonist liraglutide improves metabolic and functional recovery after cerebral ischemia in rats.

Glucagon-like peptide 1 receptor (GLP-1R) agonists administered before or immediately after induction of experimental stroke have been shown to provide acute neuroprotection. Here, we determined whether delayed treatment with a GLP-1R agonist could improve metabolic and functional recovery after stroke. Rats were subjected to middle cerebral artery occlusion (MCAO) and given the well-established GLP-1R agonist liraglutide (50, 100, or 200µg/kg) or normal saline (NS) daily for 4 weeks, starting 1 day after MCAO. Cerebral glucose metabolism and neurological deficits were evaluated using 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging and modified neurological severity score (mNSS) test. Levels of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), von Willebrand factor (vWF), and GLP-1R were assessed by immunohistochemical staining and Western blot analysis. PET imaging showed that animals treated with liraglutide had significantly higher 18F-FDG accumulation in the cerebral infarction compared with animals treated with NS. Liraglutide significantly reduced the mNSS score. It also greatly increased the expression of NeuN, GFAP, vWF, and GLP-1R in the cerebral ischemic area at postoperative week 4. These results demonstrated metabolic and functional recovery after delayed treatment with liraglutide in a rat model of cerebral ischemia.

Modulatory Effect of Fermented Papaya Extracts on Mammary Gland Hyperplasia Induced by Estrogen and Progestin in Female Rats.

Fermented papaya extracts (FPEs) are obtained by fermentation of papaya by Aspergillus oryzae and yeasts. In this study, we investigated the protective effects of FPEs on mammary gland hyperplasia induced by estrogen and progestogen. Rats were randomly divided into 6 groups, including a control group, an FPE-alone group, a model group, and three FPE treatment groups (each receiving 30, 15, or 5 ml/kg FPEs). Severe mammary gland hyperplasia was induced upon estradiol benzoate and progestin administration. FPEs could improve the pathological features of the animal model and reduce estrogen levels in the serum. Analysis of oxidant indices revealed that FPEs could increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, decrease malondialdehyde (MDA) level in the mammary glands and serum of the animal models, and decrease the proportion of cells positive for the oxidative DNA damage marker 8-oxo-dG in the mammary glands. Additionally, estradiol benzoate and progestin altered the levels of serum biochemical compounds such as aspartate transaminase (AST), total bilirubin (TBIL), and alanine transaminase (ALT), as well as hepatic oxidant indices such as SOD, GSH-Px, MDA, and 8-oxo-2'-deoxyguanosine (8-oxo-dG). These indices reverted to normal levels upon oral administration of a high dose of FPEs. Taken together, our results indicate that FPEs can protect the mammary glands and other visceral organs from oxidative damage.

Minocycline induces protective autophagy in vascular endothelial cells exposed to an in vitro model of ischemia/reperfusion-induced injury.

Minocycline has been reported to exhibit advantageous effects on ischemic stroke; however, the precise mechanism of minocycline remains to be established. In the present study, human umbilical vein endothelial cells (HUVECs) were subjected to in vitro simulated ischemia/reperfusion conditions to determine the potential effect of minocycline-induced autophagy on HUVEC damage under oxygen-glucose deprivation/reperfusion (OGD/R). The study demonstrated that minocycline enhanced autophagy in a dose-dependent manner in HUVECs exposed to OGD/R, and only low-dose minocycline protected HUVECs from OGD/R-induced damage. Subsequently, 3-methyladenine (3-MA) was added into the culture media and the protective effect of minocycline was abolished. At the same time, it has been observed that simultaneous treatment with 3-MA also inhibited the autophagy activity induced by minocycline. This finding could suggest that autophagy induced by minocycline serves as one of the potential protective mechanism underlying the beneficial effects of minocycline on ischemic injury.

[Experimental study on therapeutic effect of Dabuyin Wan on true precocious puberty in female rats].

OBJECTIVE: To study the therapeutic effect of Dabuyin Wan on true precocious puberty of female rats and its possible mechanism. METHOD: Twenty-two-day-old female SD rats were subcutaneously injected with 40 mg x kg(-1) N-methyl-DL-aspartic acid (NMA) at 14:00 and 16:00 every day; meanwhile, the rats were given Dabuyin Wan for intervention. Visual inspection was conducted for the time of vaginal opening. The first estrus was observed by yaginal smear test. Their ovaries and uterus were weighed to calculate organ coefficients. Conventional pathological slices were made to observe morphological changes in ovaries and uterus and calculate the thickness of uterine walls and the number of corpus luteums. The level of E2 in serum was detected to assess the therapeutic effect of Dabuyin Wan on NMA precocious puberty in rats. expressions of GnRH, GPR54 and Kiss-1 mRNA in hypothalamus were measured by semi-quantitative RT-PCR to investigate the possible mechanism of Dabuyin Wan. RESULT: Dabuyin Wan at 3.24 g x kg(-1) and 1.62 g x kg(-1) significantly decreased the organ coefficients in rats with precocious puberty (P < 0.05), decrease the number of vaginal openings in rats (P < 0.01) and the thickness of uterine walls and the number of corpus luteums (P < 0.05), and notably down-regulated expressions of GnRH, GPR54 and Kiss-1 mRNA in hypothalamus (P < 0.05), without significant impact on E2 in serum. CONCLUSION: Dabuyin Wan may inhibit GnRH synthesis and release as well as startup of hypothalamic-pituitary-gonadal axis by down-regulating Kiss-1/GPR54 mRNA expression in hypothalamus, in order to realize the therapeutic effect on true precocious puberty.

Effect of FTY720 on some physiological indexes of Non-Obese Diabetic (NOD) Mice.

The studies were performed to investigate the physiological characteristics of non-obese diabetic (NOD) mice treated with FTY720. At the age of 12 weeks, each mouse was fed with FTY720 or physiological saline once a day for 10 weeks running, and their blood glucose, weight, anti-GAD antibody and organ indexes were determined. No mouse in group FTY720 (NOD mice treated with FTY720) showed diabetic symptoms. The average content of serum anti-GAD antibody in group FTY720 decreased 48.75% (P < 0.01). It was concluded that the spleen, kidney and liver of NOD mice treated with FTY720 shriveled significantly in the progression of diabetes (P < 0.01 or P < 0.05). The body weight of group FTY720 mice was slightly lower than that of the model control (MC) group and these two groups both had less body weight than the normal control (NC) group (P < 0.01). The result of tests of anti-GAD antibody suggested that FTY720 treatment could suppress the anti-GAD response.

Enzymatic hydrolysis of Alaska pollack (Theragra chalcogramma) skin and antioxidant activity of the resulting hydrolysate.

BACKGROUND: Fish skin, a by-product of the food industry, contains a large amount of collagen. However, only a small proportion of fish skin is used in the production of leather materials and animal feedstuffs, most of it being discarded. The aims of this study were to prepare peptides from Alaska pollack (Theragra chalcogramma) skin by enzymatic hydrolysis and to evaluate the antioxidant activity of the resulting hydrolysate. RESULTS: Protamex was the most efficient enzyme for preparing antioxidant peptides from Alaska pollack skin. The optimal hydrolysis conditions were as follows: hydrolysis time 8 h; enzyme/substrate ratio 2:1000; skin/water ratio 1:6; temperature 55 degrees C; pH 6.0. Under these conditions the highest yield of peptides was 83.44%, with 85.95% of the hydrolysate being mainly composed of oligopeptides with molecular weights ranging from 180 to 1000 Da. The hydrolysate showed 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, with an IC(50) value of 2.5 mg mL(-1), and its reducing power was 0.14 at 1 mg mL(-1), 53.8% of that of reduced glutathione at the same concentration. CONCLUSION: This study demonstrated that the hydrolysate of Alaska pollack skin was mainly composed of oligopeptides with two to eight amino acid residues and possessed antioxidant activity.

[Protective effect of total isoflavones from Pueraria lobata on secondary osteoporosis induced by dexamethasone in rats].

OBJECTIVE: To study the protective effect of the total isoflavones from Pueraria lobata (TIP) on secondary osteoporosis induced by dexamethasome (DXM). METHODS: Female SD rats, 4 month aged, were given intramuscularly with 1.5 mg/kg DXM twice once week. The treated groups were orally given TIP 100, 50 or 25 mg.kg-1.d-1 for 6 months. The whole body BMD and BMC were detected at 3 and 6 months. The biodynamics potency, density, ash weight and calcium content of femur were determined at 6 months. RESULTS: (1) At 3 and 6 months, BMDs in TIP 100 mg/kg group were increased by 3.4% and 8.4% than that in model group, BMC were increased by 7.0% and 9.1%, while in TIP 50 mg/kg group by 3.2%, 5.3% and 6.2%, 6.0%. (2) The Fmax and hardness in TIP 100 mg/kg group were enhanced by 10.3% and 10.5%, when in TIP 50 mg/kg group were enhanced by 3.5% and 7.1%. (3) TIP can increase wet and dry weight in slightly extent, but significantly increase wet density and dry density. (4) The ash weight and calcium content in femur were increased by 3.1% and 4.2% in TIP 100 mg/kg group, 4.3% and 6.4% in TIP 50 mg/kg group, while no change in TIP 25 mg/kg group. CONCLUSIONS: TIP can prevent secondary osteoporosis induced by DXM in rats.

[Changes of element contents and their interrelationship in tibia of osteoporotic rats]

OBJECTIVE: To observe the changes of bone element contents in osteoporosis and their interrelationship. METHODS: Twelve female SD rats,10-month-old, were bilaterally ovariectomized (OVX group) and another ten rats were received sham-operation under anesthesia (SHAM group).The element contents in tibia, including Ca, P, Mg, Zn, Mn, Fe, Cu, Mo and Cr, were determined by atomic absorption spectrophotometer 7 month later. The data of contents of all elements were analyzed by simple regression. RESULTS: Compared with the SHAM group rats, the contents of Ca, P and Mg were decreased by 6.6 %(P<0.05), 6.3 %(P<0.05) and 14.9 %(P<0.01) respectively. The contents of Zn and Fe were reduced by 15.2 %(P<0.01) and 35.1 %(P<0.01) separately, Mo and Cr were decreased by 12.2 %(P>0.05) and 14.0 %(P>0.05), while the contents of Mn, Cu and Co were shown no change. There was a significant correlation among the contents of Mg, Mn, Zn, Ca and P. CONCLUSION: The contents of Ca, P, Mg, Zn and Fe were matkedly reduced in bone of osteoporotic rats induced by ovariectomy.

[Hypocholesterolemic effect of total isoflavones from Pueraria lobata in ovariectomized rats].

OBJECTIVE: To observe the effects of total isoflavones from Pueraria lobata (TIP) on lipid metabolism in estrogen deficient rat. METHODS: Female SD rats were bilaterally ovariectomized under anesthesia. TIP was administrated orally for 7 months. Serum lipids were determined at 1.5, 4.0 and 7.0 months, and liver lipids were detected at 7 months. RESULTS: Compared with non-ovariectomized rats, serum TC level and the ration of TC/HDL-C in ovariectomized rats were obviously increased while HLD-C was slightly induced and TG level were changeless. In TIP-treated groups, serum TC level and the ratios of TC/HDL-C were markedly lowered than those in OVX group, while HDL-C slightly increased. TG level were much reduced in 1.5 month and changeless in 4.0 and 7.0 month. Slightly increase of cholesterol and markedly induction of triglyceride tissue were observed in liver. CONCLUSION: TIP significantly decreased concentrations of serum TC and liver TG in OVX rats.

[Estrogen-like effects of puerarin and total isoflavones from Pueraria lobata].

OBJECTIVE: To study the estrogen-like effects of puerarin and total isoflavones from Pueraria lobata (TIP) in vivo. METHODS: Puerarin and TIP were orally administrated to ovariectomized rats, infancy or adult mice and estrogen-treated mice at the doses of 150, 300 and 600 mg/kg for 5-9 days. The estrogen-like effects were measured by viginacytology and uterus or ovary weights. RESULTS: Puerarin and TIP significantly promoted uterus growth in ovariectomized rats and infancy mice, increased the ratios of keratocytes in vaginal smear in ovariectomized rats. The sexual cycle was partially recovered in dose-dependent manner. In E2-treated mice, puerarin and TIP obviously inhibited the growth of vigina induced by E2. No obvious effect was observed in normal adult mice. CONCLUSION: The results showed that puerarin and TIP acted as weak estrogen-like effect on estrogen-deficiency animals, while no effect on normal-estrogen level ones, but as antiestrogen-like effect in high-estrogen-level ones. These results suggested that puerarin and TIP possessed property of partial agonist of estrogen receptor.