AGRICULTURAL RESEARCH COMMUNICATION CENTRE

www.arccjournals.com / www.ijaronline.inIndian J. Anim. Res., 48 (5) : 464-468, 2014

doi:10.5958/0976-0555.2014.00012.0

PROTECTION OF SALVIA MILTIORRHIZA AGAINST ACUTESODIUM NITRITE POISONING IN MICE

Xiaomei Lu* , Zhijie Lai and Meng jia Du

Department of Pathophysiology,College of Basic Medical Sciences, China Medical University, ShenYang, 110 001, China

Received: 17-02-2014 Accepted: 14-04-2014

ABSTRACTThe present study evaluated the protection of Salvia miltiorrhiza injection against acute sodium

nitrite poisoning in mice. Forty male C57mice were randomly divided into control group, sodiumnitrite poisoning group (SN), Methylene blue (MB) and Salvia miltiorrhizae (SM) group. In SN, MBand SM group, 200 mg/kg sodium nitrite was injected intraperitoneal. Mice in MB and SM groupwere given 1.5mg/kg Methylene blue and 0.2ml Salvia miltiorrhiza injection immediately after sodiumnitrite injection respectively. Mice in SN and SM group demonstrated higher blood methemoglobinand Serum TNFá and IL-6 but lower IL-10 levels than control group (P< 0.05). HE and TUNELstaining showed developed histological damage accompanied with increased apoptosis in heart,lung, liver and kidney. But the values were much less severe in MB and SM group than those in SNgroup(P< 0.05).The results of this study showed that Salvia miltiorrhizae injection reduced tissueinjury induced by sodium nitrite effectively.

Key words: Mice, Salvia miltiorrhiza, Sodium nitrite.

*Corresponding author’s e-mail: luxmei@hotmail.com

INTRODUCTIONSodium nitrite is a white to slight yellowish

crystalline powder that is very soluble in water andis hygroscopic( Hassan et al., 2010). It is a usefulprecursor to a variety of organic compounds, suchas pharmaceuticals, dyes, and pesticides, but it isprobably best known as a food additive to preventbotulism( Luca et al.,1987). While this chemical willprevent the growth of bacteria, it can be toxic inhigh amounts for animals, including humans. If oneis eating much sodium nitrite, methemoglobinemiawil l happen( Gautami et al.,1995).Methemoglobinemia is a disorder characterized bythe presence of a higher than normal level ofmethemoglobin in the blood. Methemoglobin is aform of hemoglobin that contains ferric (Fe3+ ) ironand has a decreased ability to bind oxygen. Whenmethemoglobin concentration is elevated in redblood cells, tissue hypoxia can occur ( Brown et al.,2013). Signs and symptoms of methemoglobinemiainclude shortness of breath, cyanosis is the classicsymptom, mental status changes, headache, fatigue,exercise intolerance, dizziness and loss ofconsciousness. Arterial blood with elevated

methemoglobin levels has a characteristic chocolate-brown color as compared to normal bright redoxygen-containing arterial blood( Hall et al., 2013).Methemoglobinemia can be treated wi thsupplemental oxygen and methylene blue. Methyleneblue restores the iron in hemoglobin to its normal(reduced) oxygen-carrying state(Singh et al., 2012).But at high doses, however, methylene blue actuallyinduces methemoglobinemia.

Traditional Chinese medicine has evolvedinto a well-developed, coherent system of medicineto treat illness( Ni et al., 2012). Herbal medicinehas been used for more than 2000 years ( Zheng etal., 2011). Salvia miltiorrhiza is a traditional Chinesedrug commonly used for activating circulation( Hanet al., 2008), the research on the eûect of it in thetreatment of sodium nitrite poisoning has not beenreported. Salvia miltiorrhiza injection is theextraction of wild Salvia roots, the main activeingredients include danshensu, salvianolic acid, aswell as tanshinone, dihydrotanshinone, andcryptotanshinone(Yu et al., 2012), which are ableto protect endothelial cells, fight againstinûammation, and prevent lipid peroxidation and

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calcium overload( Zhao et al., 2004; Xia et al., 2003;Chen et al., 2006) .Some studies have shown thatSalvia miltiorrhiza is used to treat some kind ofpoisoning such as mercury poisoning(Jing et al.,2010) and carbon tetrachloride poisoning(Wang etal., 2012) through protecting multi organ functions.Although salvia miltiorrhiza has been used to treatsome kind of poisoning, the exact mechanism is notvery clear. For this reason, the purpose of this studyis to evaluate the protection of salvia miltiorrhizainjection against acute sodium nitrite poisoning inmice through observing the pathological alterationsin the heart, lung, liver and kidney tissues.

MATERIALS AND METHODSAnimals: Eight to ten weeks old male C57BL/6 miceweighing 20–25 g were obtained from theExperimental Animal Center of China MedicalUniversity. All were fed a standard laboratory chowand allowed tap water ad libitum All the experimentalprocedures were performed according to theguidelines for the care and use of animals establishedby China Medical University. Fourty age-matchedmale C57mice were randomly divided into controlgroup, sodium nitrite poisoning group (SN),Methylene blue (MB) and Salvia miltiorrhizae-treated(SM) group. In SN, MB and SM group, 200mg/kg sodium nitrite was injected intraperitoneal.Mice in MB and SM group were given 1.5mg/kgMethylene blue and 0.2ml Salvia miltiorrhizainject ion(each 10mL vial contains activecomponents equivalent to 15g of the originalmedicine, Shenghe Pharmaceutical Co., Ltd.China.) immediately after sodium nitrite injectionrespectively. In control group, an equal volume ofsterile saline was administered. All the mice weresacrificed after 24 h of sodium nitrite injectionÿBloodsamples and heart, lung, liver and kidney tissues werecollected while they were under anesthesia.

Methemoglobin analysis: After 24h of sodiumnitrite injection , the blood was collected for themeasurement of methemoglobin analysis. Firsthemoglobin content was determined by hemoglobindetermination reagent, then methemoglobin wasdetermined by methemoglobin determinationreagent. And then methemoglobin percentage wascalculated according to the manufacturer’sinstructions. (Nanjing Jiancheng,China).

Observation of mortality rate and pathologicalchanges: After 24h of Sodium nitrite injection, themortality rates of mice in various groups wererecorded and the animals were killed by cervicaldislocation, followed by immediate organ collectionfor histologic analysis. Fresh heart, lung, liver andkidney tissues sections were fixed in 10% bufferedformalin and embedded in paraffin; 4 ìm sectionswere stained with hematoxylin and eosin. Apathologist blind to group assignment analyzed thesamples and determined the levels of injury.

Determination of apoptosis: Detect ion ofapoptotic cells was carried out using the terminaldeoxynucleotidyl transferase-mediated dUTPnickend labeling (TUNEL) assay kit. Fresh heart,lung, liver and kidney sections were fixed in 10%buffered formalin and embedded in paraffin; 4 ìmthick of slices were stained using the in situ Cell DeathDetection kit (Promega technology, USA) accordingto the manufacturer’s instructions. Three sectionsfrom each sample were randomly selected and tenmicroscopic fields per section were evaluated by twoindependent observers. In each field, nuclei werecounted and the percentage of TUNEL-positivenuclei was calculated.

Measurement of concentrations of cytokinesin serum: The concentrations of TNF- a, IL-6 andIL-10 in serum were measured by using commercialavailable ELISA kits (Ray Biotech, USA) followingthe manufacturer’s instructions. They were carefullychecked for specify, sensitivity and reliability. Serumconcentration of TNF- a, IL-6 and IL-10 weremeasured using mouse ELISA kits: ELM-TNFalpha-001, ELM-IL6-001 and ELM-IL10-001 respectively.The absorbance was measured using aspectrophotometer (Biotek, USA); concentration ofeach cytokine was calculated by a comparison curveestablished in the same measurement using prism 5Graphpad. Each cytokine assay was performed induplicate each time.

Statistical analysis: All values presented as means± SE. Differences were compared by ANOVAfollowed by Bonferroni correction for post hoc t-test,where appropriate. Probabilities of < 0.05 wereconsidered to be statistically significant. All of thestatistical tests were performed with the SPSSsoftware 13.0.

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TABLE 1: Methemoglobin concentration in each group (x± s, n= 6).

Methemoglobin concentration (g/l) Methemoglobin percentage (%)

CONTROL group 7.8± 1.9 12.6± 2.8Sodium nitrite group 29.9± 2.3* 40.4± 3.5*methylene blue-treated group 12.8± 1.9*# 17.8± 2.8*#Salvia miltiorrhizae-treated group 29.8± 1.8* 38.6± 3.3*

Note: ÿP< 0.05 vs Control group, ÿP< 0.05 vs Sodium nitrite groupÿ

CON SN MB SM

RESULTS AND DISCUSSIONComparison of mortality rates: Four mice diedin the sodium nitrite group; 2 mice died in SM groupand no mice died in the MB group and control group.

Methemoglobin analysis: Sodium Nitrite inducedincrease of methemoglobin levels compared withcontrol group(P < 0.05), however, in MB and SMgroup was significant lower than in SN group (P <0.05). As illustrated in Table 1.

Histological changes of heart, lung, liver andkidney tissues: Tissues sections were stained withhematoxylin and eosin. The results were shown inFigure 1. From this figure it can be seen that in controlgroup, there is no obvious abnormality under thelight microscope. While in the SN group, swellingand necrosis of tissues and inûammatory cellinûltration and edema of connective tissue wereseen. In MB and SM Group, which was lighter thanthat in SN group.

Figure 1. Heart, lung, liver and kidney sections werestained with hematoxylin and eosin. A-D meansheart tissue, E-H means lung tissue, I-L means livertissue and M-P means kidney tissue. CON meanscontrol group, SN means sodium nitrite poisoninggroup, MB means methylene blue and SM means

Salvia miltiorrhizae-treated group.There is noobvious abnormality in control group. While in theSN group, swelling and necrosis and inûammatorycell inûltration and edema of connective tissue wereseen. In SM group and MB group, which was lighterthan that in SN group.

Apoptosis in renal tissue:Sodium nitrite inducedincrease of TUNEL staining compared with controlgroup (P < 0.05), however, these increase in MBand SM group was significant lower than in SNgroup (P < 0.05). As illustrated in Figure 2.

Figure 2. TUNEL staining to show the tissueapoptosis. A-D means heart tissue, E-H means lungtissue, I-L means liver tissue and M-P means kidneytissue. CON means control group, SN meanssodium nitri te poisoning group, MB meansmethylene blue and SM means Salvia miltiorrhizae-treated group. Sodium nitrite induced increase ofTUNEL staining compared with control group,however, these increase in SM group and MB groupwas significant lower than in Sodium nitrite group.

Inflammatory cytokines release in serum:Sodium nitrite induced increase of TNF-± and IL-6release and decrease of IL-10 compared with control

CON SN MB SM

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TABLE 2: TNF-a, IL-6 and IL-10 concentration in serum (x± s, n= 8).

TNF-± (pg/µg protein) IL-6(pg/µg protein) IL-10(pg/µg protein)

CONTROL group 13.8± 0.9 11.3± 0.8 10.4± 0.7Sodium nitrite group 19.5± 1.9* 18.8± 1.5ÿ 3.7± 0.4*Methylene blue-treated group 14.3± 1.2*# 13.6± 1.4ÿ 9.4± 0.9*#Salvia miltiorrhizae-treated group 15.4± 0.7* 15.6± 1.3ÿ 8.3± 0.6*

Note: ÿP< 0.05 vs Control group, ÿP< 0.05 vs Sodium nitrite groupÿ

group(P < 0.05), however, TNF-± and IL-6 in MBand SM group was significant lower and IL-10significantly higher than in SN group (P < 0.05). Asillustrated in table 2.

Sodium nitrite is widely used in industry,construction and meat products. Nitrite is highlytoxic substances, adult intake of 0.2 to 0.5 g cancause poisoning, 3 grams can be lethal( Luca etal., 1987). Due to the consumption of high levelsof nitrate or nitrite cured meat, pickles, and thespoiled vegetables can cause poisoning ( Lukin-Butenko et al., 1983) . Nitrite will trigger oxidationof low iron in the normal oxygen-carryinghemoglobin into methemoglobin, thus losing theoxygen-carrying capacity and cause t issuehypoxia( Gautami et al., 1995). Nitrite is also acarcinogen, according to the previous studies,esophageal cancer patients was posi t ivelycorrelated with nitrite salt intake ( Xie et al., 2011).Nitrite poisoning incidence rapid incubationperiod is generally one to three hours, the mainfeatures of the poisoning is due to tissue hypoxiacaused cyanosis phenomenon, such as the lips,tongue and body skin bruising. Sleepiness ori r ri tabi l i ty, d i f f iculty breathing, d izziness,headache, fatigue, respiratory failure and death(Gautami et al., 1995). In our study, 4 mice diedin the sodium nitrite group, and after sodiumnitrite treatment, all the extremities and the tip ofthe nose, skin and mucous membrane of micecyanosis, the mice showed incontinence anddecreased activities. Which means we successfullyestablished nitrite poisoning animal model andpresented methemoglobinemia obviously.Methemoglobinemia can be treated wi thsupplemental oxygen and methylene blue.Methylene blue restores the iron in hemoglobin toits normal (reduced) oxygen-carrying state. Thisis achieved by providing an artificial electron

acceptor for NADPH methemoglobin reductase .At high doses, however, methylene blue actuallyinduces methemoglobinemia, Salvia miltiorrhiza inthe experiment, as an old Chinese herb, was shownto be valuable for many kinds of disease previously.All these research reveal that Salvia miltiorrhiza playa role in the treatment of some kind of disease by itsproperties such as protect endothelial cells, ûghtagainst inûammation, and prevent lipid peroxidation. In our study, we used the animal model to mimicthe sodium nitrite poisoning in humans bothbiochemically and morphologically. Thecharacteristic histological features of our modelswere findings of swelling and necrosis of tissues ,inûammatory cell inûltration and edema ofconnective tissue in interstitium. And the model alsoshown an increase of methemoglobin , accompaniedwith increase of inflammatory cytokines andapoptosis in tissues. In the study, we observed thatSalvia miltiorrhiza attenuated the histologic severityof tissues. So it can be speculated on the mechanismof Salvia miltiorrhiza on experimental sodium nitritepoisoning. It can prevent the inflammation andapoptosis in tissue. However, there is still a greatneed for more and better research on the efficacyand safety of Salvia miltiorrhiza. Although it is difficultto extrapolate animal data to humans, best Salviamiltiorrhiza is recommendedas a beneficial treatmentfor sodium nitrite poisoning.

CONCLUSIONSSalvia miltiorrhizae injection reduced tissue

injury induced by Sodium nitrite effectively, whichmay served as a beneficial treatment accompaniedwith methylene blue to rescue acute sodium nitritepoisoning patients.

ACKNOWLEDGMENTSThis study was supported in part by the

grants from the National Natural Science Foundationof China (NO.81100109).

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