Picroside II attenuates ischemia/reperfusion testicular injury by alleviating oxidative stress and apoptosis through reducing nitric oxide synthesis

Abstract Purpose: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. Methods: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. Results: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). Conclusion: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.

Investigation of Decoction Process of Digda-4 Decoction / 中国实验方剂学杂志

Objective:To optimize the decoction process of Digda-4 decoction(DGD-4D), and provide reference for the standardization study of decoction of Mongolian medicine decoction. Method:Taking DGD-4D as model drug, different decoction methods of Mongolian medicine were compared, HPLC was used to determine contents of aesculetin, geniposide, picroside Ⅰ and picroside Ⅱ.On the basis of single factor tests, central composite design-response surface methodology was adopted to optimize the decoction process of DGD-4D with transfer rates of 4 components and dry extract rate as indexes, regression model fitting was carried out by Design-Expert 8.0.6 software, prediction model of process parameters was established, and the optimal process was verified. Result:The optimal decoction condition of DGD-4D was determined to be adding 40 times the amount of water and decocting for 17 min, decocting once.Transfer rates of aesculetin, geniposide, picroside Ⅰ, picroside Ⅱ and dry extract rate were 70.01%, 94.11%, 61.23%, 92.32%, 32.89%, respectively. Conclusion:The optimum decoction process of DGD-4D is established, it has important reference significance for excavating, sorting, improving the level of Mongolian medicine preparations and ensuring the consistency of their clinical efficacy.

Effect of Picroside Ⅱ on Expression of microRNA-1 in H2O2-induced H9c2 Cardiomyocytes Damage / 中国实验方剂学杂志

Objective: To study the effect of picroside Ⅱ on the expression of microRNA-1 (miR-1) in the H2O2-induced H9c2 cardiomyocytes damage, in order to explore the mechanism of picroside Ⅱ in protecting H9c2 cardiomyocytes from oxidative stress. Method: H9c2 cardiomyocytes were divided into 6 groups:control group, model group (H2O2 200 μmol·L-1), picroside Ⅱ (50, 100, 200 μmol·L-1)+H2O2 (200 μmol·L-1) group and picroside Ⅱ (200 μmol·L-1) group. Picroside Ⅱ group was incubated with picroside Ⅱ for 6 h and then cultured with H2O2 for 2 h. At the end of drugs treatment, the cell viability and the cellular damage of cardiomyocytes were respectively assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. 4',6-diamidino-2-phenylindole (DAPI) staining and cysteinyl aspartate specific proteinase-3 (Caspase-3) test were used to evaluate cell apoptosis. The mRNA expressions of Caspase-3,B-cell lymphoma-2 (Bcl-2) and miR-1 were measured by Real-time polymerase chain reaction (Real-time PCR). The protein expression of Bcl-2 was detected by Western blot. Result:Compared with the control group, H2O2 could significantly decrease the cell viability and increase the rate of apoptosis, up-regulate mRNA expression of Caspase-3 and miR-1, and down-regulate expression of Bcl-2 in H9c2 cells (PPPPPPPConclusion:Picroside Ⅱ has a protective effect on H9c2 cells from H2O2-induced cardiomyocyte injury by down-regulating mRNA-1 expression and up-regulating the expression of the downstream Bcl-2.

Picroside II attenuates fatty acid accumulation in HepG2 cells via modulation of fatty acid uptake and synthesis

BACKGROUND/AIMS: Hepatic steatosis is caused by an imbalance between free fatty acids (FFAs) uptake, utilization, storage, and disposal. Understanding the molecular mechanisms involved in FFAs accumulation and its modulation could drive the development of potential therapies for Nonalcoholic fatty liver disease. The aim of the current study was to explore the effects of picroside II, a phytoactive found in Picrorhiza kurroa, on fatty acid accumulation vis-à-vis silibinin, a known hepatoprotective phytoactive from Silybum marianum. METHODS: HepG2 cells were loaded with FFAs (oleic acid:palmitic acid/2:1) for 20 hours to mimic hepatic steatosis. The FFAs concentration achieving maximum fat accumulation and minimal cytotoxicity (500 μM) was standardized. HepG2 cells were exposed to the standardized FFAs concentration with and without picroside II pretreatment. RESULTS: Picroside II pretreatment inhibited FFAs-induced lipid accumulation by attenuating the expression of fatty acid transport protein 5, sterol regulatory element binding protein 1 and stearoyl CoA desaturase. Preatreatment with picroside II was also found to decrease the expression of forkhead box protein O1 and phosphoenolpyruvate carboxykinase. CONCLUSIONS: These findings suggest that picroside II effectively attenuated fatty acid accumulation by decreasing FFAs uptake and lipogenesis. Picroside II also decreased the expression of gluconeogenic genes.

Picroside Ⅱ plays a neuroprotective effect by inhibiting cyto C/caspase-9/caspase-3 signal pathway following ischemia/reperfusion injury in rats / 中国药理学通报

Aim To investigate the neuroprotective effect of picroside Ⅱ(PIC)on cyto C/caspase-9/caspase-3 signal pathway following ischemia/reperfusion(I/R)injury in rats.Methods Atractyloside(Atr)was selected as negative control,cyclosporin A(CsA)was selected as positive control,and PIC was selected as the treatment medicine.The I/R model was made by inserting a monofilament suture into internal carotid artery for 2 h,and then reperfused for 24 h.The cerebral infarction volume was detected by TTC staining,and the expression of cyto C,caspase-9 and caspase-3 were determined by immunohistochemical assay and Western blot.Results In model group,the cerebral infarct volume was obviously large;the expression of cyto C,caspase-9 and caspase-3 was increased significantly more than that in sham group(P<0.05).In PIC group,the cerebral infarct volume was significantly improved;the expression of cyto C,caspase-9 and caspase-3 was significantly decreased than that in model group(P<0.05).In Atr+PIC group,the rat infarction volume was reduced,and the expression of cyto C,caspase-9 and caspase-3 was significantly decreased than that in Atr group(P<0.05).Conclusion The mechanism of PIC inhibiting neuron apoptosis in focal cerebral I/R rats might be through down-regulating the expression of cyto C,caspase-9 and caspase-3.

The effect of picroside II on the ERK1/2 signal transduction pathway and its neuroprotective effect on the cerebral ischemic injury in rats / 中华行为医学与脑科学杂志

Objective To explore the neuroprotective effect and mechanism of picroside II on ERK1/2 signal transduction pathway after cerebral ischemia injury in rats.Methods The focal cerebral is-chemic models were established by inserting a monofilament threads into middle cerebral artery occlusion (MCAO) in 100 Wistar rats and treated by injecting picroside II (20 mg/kg) intraperitoneally.The neu-robehavioral function was evaluated by modified neurological severity score points ( mNSS) test.The cerebral infarct volume was measured by tetrazolium chloride ( TTC) staining.The apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL) assay.The expression of pERK1/2 in cortex was determined by the immunohistochemistry ( IHC) and Western Blot ( WB) .Results mNSS test showed that severe neurological dysfunction was found in model and LPS groups,and the scores of mNSS were significantly increased;meanwhile the scores of mNSS in treatment group and U0126 group were signifi-cantly lower than that in model and LPS groups (P<0.05).TUNEL assay showed that the apoptotic cell inde-xes (ACI) in different groups were (0.06±0.02),(0.27±0.03),(0.07±0.02),(0.26±0.03)and(0.09± 0.05) ,and the ACI in treatment and U0126 groups was obviously lower than that in model and LPS groups (P<0.05) .With IHC and WB,pERK1/2 level in model group was the highest,which was slightly higher than that of LPS group,and pERK1/2 expression in treatment and U0126 groups was significantly decreased com-pared with that in model and LPS groups (P<0.05) .Conclusion The activation of ERK1/2 by cerebral is-chemia could induce the cell apoptosis.Picroside II might reduce cell apoptosis by inhibiting the activation of ERK1/2 in ischemic brain injury.

Residual Determination of 7 Organic Solvents in PicrosideⅡRaw Material by Head-space GC / 中国药房

OBJECTIVE:To establish the method for the residual determination of 7 organic solvents in picrosideⅡraw materi-als. METHODS:Head-space GC was performed on the capillary column of 6% cyanopropyl phenyl-94% dimethyl polysiloxane (DB-624) by temperature programming,the temperature of injector was 200 ℃,temperature of flame ionization detector was 250 ℃,the flow rate of N2 was 35 ml/min,and split ration was 10∶1,headspace sampling was adopted with the volume of 1 ml, the heating temperature of headspace sampling was 85 ℃,heating time was 45 min. RESULTS:The good linear relationship of methanol,ethanol,ethylacetate,methylbenzene,benzene,phenylethylene and divinglbenzene had been obtained(r=0.999 6-0.999 9);RSDs of precision stability test were less than 3%;average recoveries was in the range of 78.0%-104.9%(RSDs were 0.65%-2.47%,n=6)respectively. CONCLUSIONS:The method is specific,rapid,simple and accurate,and can be used for the determination of residual organic solvents in picrosideⅡraw materials.

Effect of picrosideⅡon expression of myelin basic protein after cerebral ischemia injury in rats / 中国病理生理杂志

AIM: To verify the neuroprotective effect and optimize the therapeutic dose and time window of picroside Ⅱon cerebral ischemic injury in rats .METHODS:The forebrain ischemia model was established by the method of bilateral common carotid artery occlusion ( BCCAO ) .The successful model rats were randomly divided into 16 groups according to orthogonal design and treated by intraperitoneal injection of picroside Ⅱat different ischemic time poinis and different doses .The changes of the nerve fiber myelin were observed by fast green staining .The immunohistochemical assay and Western blotting were used to quantitatively and qualitatively determine the expression of myelin basic protein (MBP). The mRNA level of MBP in the brain tissues was tested by reverse transcription polymerase chain reaction (RT-PCR).RE-SULTS:Picroside Ⅱ increased the expression of MBP and decreased demyelination after cerebral ischemic injury .The best therapeutic time window and dose were:(1) ischemia for 2.0 h with picrosideⅡat dose of 10 mg/kg according to the results of fast green staining;(2) ischemia for 2.0 h with the dose of 10 mg/kg according to the results of immunohisto-chemical assay;(3) ischemia for 2.0 h with the dose of 10 mg/kg according to the analysis of Western blotting;(4) is-chemia for 1.5 h with the dose of 20 mg/kg according to the detection of RT-PCR.CONCLUSION:Given the principle of the lowest therapeutic dose with the longest time window , the optimized therapeutic dose and time window for rat cerebral ischemic injury is intraperitoneal injection of picroside Ⅱat the doses of 10~20 mg/kg and the time window of ischemia for 1.5~2.0 h.

Antioxidation of Picroside Ⅱ on Cerebral Ischemic Injury in Rats and Its Optimum Dosage and Time Window / 中国中医药信息杂志

Objective To optimize the therapeutic dose and time window of picroside Ⅱ in treating cerebral ischemic injury in rats by orthogonal test. Methods The forebrain ischemia models were established by bilateral common carotid artery occlusion (BCCAO) method. The successful models were randomly grouped according to orthogonal experimental design and treated by injecting picroside Ⅱintraperitoneally at different ischemic time with different doses. The concentrations of MDA, NO and H2O2 in serum and brain tissue were respectively determined by thiobarbituric acid assay, nitratase reductase assay and chemiluminescence immunoassay. Results The optimized composition of the therapeutic dose and time window of picroside Ⅱ in cerebral ischemic injury were ischemia 1.5 h with 10 mg/kg, 1.5 h with 20 mg/kg and 1.5 h with 10 mg/kg body weight according to the expressions of MDA, NO and H2O2 in serum, and ischemia 1.5 h with 10 mg/kg, 1.5 h with 20 mg/kg and 1.5 h with 20 mg/kg body weight according to the expressions of MDA, NO and H2O2 in brain tissue. Conclusion On the basis of the principle of lowest therapeutic dose with longest time window, the optimized composition of the therapeutic dose and time window in cerebral ischemic injury is injecting picroside Ⅱ intraperitoneally with 10-20 mg/kg body weight at ischemia 1.5 h.

The anti-oxidant effect and the possible mechanism of plcroside Ⅱ in cerebral ischemia/reperfusion injury in rats / 国际中医中药杂志

Objective To investigate the anti-oxidant effect and the possible mechanisms ofpicrodide II in cerebral ischemia/reperfusion injuries in rats. Methods A total of 90 adult, healthy, mmale Wistar rats were used to established the middle cerebral artery occlusion reperfusion (MCAO/R) models by intraluminal monofilament suture on the left external-internal carotid artery. The treatment group and the positive control group were respectively injected with 1.0％ picroside II (10 mg/kg, 250 μl) and salvianic acid A sodium (10 mg/kg, 250 μl) via the tail vein, and the negative control group and sham-surgery group were injected with 0.1mol/L phosphate buffer saline (PBS) 250 μl. The neurological deficit scores were evaluated with Bederson's test. The cerebral infarction volume was observed with tetrazolium (TTC) staining. The apoptosis positive cells were counted by terminal deoxynucleotidyl transferase dUTP nick-end labeling and the expressions of inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) were detected with immunohistochemical assay.The concentration of iNOS and SOD proteins in brain tissue was detected by enzyme linked immunosorbent assay.Results Neurological behavioral malfunction appeared in all the rats with MCAO/R. The infarction focuses emerged in the ischemic hemisphere following the MCAO/R injuries. The number of apoptotic cells and the expression of iNOS increased while the SOD reduced after MCAO/R. After the treatment of picrodide Ⅱ, the nervous behavioral function (1.28±0.38)improved, the infarction volume(68.73±4.46)％ reduced, the number of apoptosis positive cells(6.10± 1.26), the expressions and the concentrations in brain tissue of iNOS(4.67+0.51)decressed while those of SOD (0.53 ±0.14) increased significantly compared with the negative control groups(t=3.16、 2.51、 4.15、3.12、 3.25, P＜0.05). Conclusion PicrodideⅡ might play a neuroprotective effect by inhibiting the neuronal apoptosis and the expressions of iNOS and SOD after cerebral ischemia/reperfusion injuries.

Online HPLC-DPPH method for antioxidant activity of Picrorhiza kurroa Royle ex Benth. and characterization of kutkoside by Ultra-Performance LC-electrospray ionization quadrupole time-of-flight mass spectrometry.

Picrorhiza kurroa Royle ex Benth., is widely used in the Indian systems of medicine for the treatment of various liver ailments. Since, the role of oxi­dative stress in the pathogenesis of liver injury has become generally recognized, in present study the free radical scavenging effect of P. kurroa was assessed by on-line HPLC-DPPH and colorimetric DPPH methods. The comparative study on antioxidant activity of P. kurroa extracts by both methods revealed that colorimetric method showed very less free radical scavenging effect while HPLC-DPPH method showed high activity. Further, the kutkoside, an important ingredient of a potent hepatoprotective formulation “kutkin/ picroliv” was investigated for its chemical composition by ultra-performance liquid chromatography coupled with diode array detection/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-DAD/ESI-QTOF-MS). Kutkoside was considered to be a single compound and reported as picroside-II or kutkoside, however, present investigation illustrated that kutkoside is a mixture of iridoid glycosides namely, picroside II, picroside IV and 6-ferulloylcatalpol.

The interferring effects of picrosideⅡ on the expressions of NF-κB and I-κB following cerebral ischemia reperfusion injury in rats / 中国药理学通报

Aim To study the interfering effects of picrosideⅡ on the expressions of nuclear transcription factor kappaB(NF-κB)and inhibitor of NF-κB(I-κB)after cerebral ischemic reperfusion in rats.Methods Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in rats.PicrosideⅡ(10 mg·kg~(-1))and salvianic acid A sodium(10 mg·kg~(-1))were injected from the tail vein for treatment.TUNEL positive cells were counted by immunofluorescence assay.The expressions of NF-κB and I-κB were determined by immunohistochemical assay,and the concentration of NF-κB and I-κB in brain tissue was determined by ELISA.Results The exprssions of NF-κB and I-κB were weakly and the apoptotic cells were scattering at cortex,striatum and hippocampus in the sham operative group.In the negative control group,the number of TUNEL positive cells and the expressions of NF-κB and I-κB increased,the absorption(A)values and the concentration were significantly higher than those in the sham operative group(P<0.05).While in the positive control and picroside groups,the expressions(A values)and concentration of NF-κB and I-κB and the number of TUNEL positive cells were significantly lower than those in the negative control group(P<0.05).There was no significant difference between the positive control group and picroside group(P>0.05).Conclusion Picroside Ⅱ might downregulate the expressions of NF-κB and I-κB to inhibit neuronal apoptosis induced by inflammation after cerebral ischemia reperfusion injury in rats.

The interference of picrosideⅡ on the expressions of Caspase-3 and PARP following cerebral ischemia reperfusion injury in rats / 中国药理学通报

Aim To explore the effect of picrodideⅡ on the expressions of Caspase-3 and poly ADP-ribose polymerase (PARP) in brain tissue following cerebral ischemic reperfusion injury in rats.Methods The middle cerebral artery occlusion reperfusion models were established with intraluminal thread methods in rats. PicrodideⅡ (10 mg·kg~(-1)) and salvianic acid A sodium (10 mg·kg~(-1)) were injected from tail vein for treatment. The neurological function was evaluated with Bederson's test and the cerebral infarction volume was observed with tetrazolium chloride (TTC) staining.The brain structure was observed by hematoxylin-eosin (HE) staining and the apoptosis was counted by TUNEL immunofluorescence assay. The expressions of Caspase-3 and PARP were detected with immunohistochemical and enzyme linked immunosorbent assay.Results After ischemia 2 h and reperfusion 22 h, the rats showed neurological function deficit and cerebral infarction in ischemic hemisphere. The expressions of Caspase-3 and PARP and the number of apoptotic cells in brain tissue increased compared with those in the sham operative group (P <0.05). In picroside and salvianic acid A sodium groups, the Bederson's scores and cerebral infarction volume, the expressions of Caspase-3 and PARP and the number of apoptosis cells were lower than those in the negative control group (P <0.05). While there was no significant difference in five indexes metioned above between picroside group and salvianic acid A sodium group (P >0.05).Conclusion PicrosideⅡ might reduce the expressions of Caspase-3 and PARP to inhibit the neuronal apoptosis induced by cerebral ischemia reperfusion injury and improve the neurological function of rats.

The interference of picroside Ⅱ on the expressions of Caspase-3 following cerebral ischemia reperfusion injury in rats / 国际中医中药杂志

Objective To explore the effect of picrodideⅡ on the expressions of Caspase-3 in brain tissue following cerebral ischemic reperfusion injury in rats. Methods The middle cerebral artery occlusion reperfusion models were established with intraluminal thread methods in rats. Picrodide Ⅱ (10 mg/kg) was injected from tail vein for treatment. The neurological function was evaluated with Bederson's test. The brain structure was observed by hematoxylin-eosin (HE)stain and the apoptosis were counted by TUNEL immunofiuorescence assay. The expressions of Caspase-3 were detected with immunohistochemical and enzyme linked immunosorbent assay. Results After ischemia for 2 h and reperfusion for 22 h, the rats showed neurological function deficit. The expressions of Caspase-3 and the number of apoptotic cells in brain tissue increased significantly than those in the sham operative group (P＜ 0.01). In picroside, the Bederson's scores, the expressions of Caspase-3 and the number of apoptosis cells were significantly lower than those in the negative control group(P＜0.01).Conclusion Picroside Ⅱ might reduce the expressions of Caspase-3 to inhibit the neuronal apoptosis induced by cerebral ischemia reperfusion injury and improve the neurological function of rats.

Synergistic protective effect of picroside Ⅱ and NGF on PC12 cells against oxidative stress induced by H2O2 / 中国临床药理学与治疗学

AIM: To study the synergistic protective effect of picrosideII and NGF for the oxidative stress on PC12 cells induced by hydrogen peroxide (H2O2). METHODS: The fluorescent probe 6-carboxy-2',7'-dichlorodihydrofluorescein (CDCFH) was used to assess the intracellular reactiveoxygen species (ROS) level, and MTT assay, morphological observation as well aslactate dehydrogenase (LDH) leakage were conducted to measure cellular injury. RESULTS: The H2O2-induced cytotoxicity was significantly attenuated in the presence of picroside II (25 μg/mL) and NGF (2 ng/mL). Cultures with this combined treatment possessed decreased level of ROS while increased cell survival, as compared to that of picroside II or NGF alone-treated cells. Accordingly, it was concluded that their synergistic protective activities against oxidative stress in vitro were demonstrated in various aspects including reversing morphological changes, enhancingthe ability of cell proliferation and ROS scavenging. CONCLUSION: Such action supports the therapeutic potential of picroside II and NGF in treating nervous disorders based on their synergistic effect.

The interferring effects of picroside Ⅱ on the expressions of NF-?B and I-?B following cerebral ischemia reperfusion injury in rats / 中国药理学通报

Aim To study the interfering effects of picroside Ⅱ on the expressions of nuclear transcription factor kappaB(NF-?B)and inhibitor of NF-?B(I-?B) after cerebral ischemic reperfusion in rats. Methods Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in rats. PicrosideⅡ(10 mg?kg-1) and salvianic acid A sodium(10 mg?kg-1 ) were injected from the tail vein for treatment. TUNEL positive cells were counted by immunofluorescence assay. The expressions of NF-?B and I-?B were determined by immunohistochemical assay,and the concentration of NF-?B and I-?B in brain tissue was determined by ELISA. Results The exprssions of NF-?B and I-?B were weakly and the apoptotic cells were scattering at cortex,striatum and hip-pocampus in the sham operative group. In the negative control group,the number of TUNEL positive cells and the expressions of NF-?B and I-?B increased,the absorption(A) values and the concentration were significantly higher than those in the sham operative group(P0.05 ). Conclusion Picroside Ⅱ might downregulate the expressions of NF-?B and I-?B to inhibit neuronal apoptosis induced by inflammation after cerebral ischemia reperfusion injury in rats.