Effects of Fufang Shenhua Tablet(复方肾华片) on the Expression of Toll-Like Receptors during Acute Kidney Injury Induced by Ischemia-Reperfusion in Rats

<正>Objective:To investigate the impact of a traditional Chinese medicinal compound known as Fufang Shenhua Tablet(复方肾华片,SHP) on the expression of Toll-like receptors(TLRs) during renal ischemia-reperfusion injury(IRI)-induced acute kidney injury(AKI) in rats.Methods:A total of 28 Wistar rats were randomly divided into five groups:(1) pseudo-operation control group,(2) ischemia-reperfusion model group, (3) Astragaloside group,(4) high-dose SHP group,and(5) low-dose SHP group.There were four rats in the pseudo-operation group and six rats in each of the other groups.The accepted ischemia-reperfusion model was established after a 7-day gavage intervention,and pathological changes and renal function were observed,using an enzyme-linked immunosorbent assay(ELISA) to detect interleukin 8(IL-8) and interferon gamma(IFN-γ) levels,as well as immunohistochemical staining to detect altered levels of TLR2 and TLR4 expression in renal tissue.Results:After 24 h,renal pathological damage and the expression levels of serum creatinine(Scr),IL-8, IFN-γ,TLR2,and TLR4 were significantly higher in the model group as compared with the pseudo-operation group(P0.05).In addition,at 24 h the above indicators decreased significantly in the Astragaloside group,high-dose SHP group and low-dose SHP group as compared with the ischemia-reperfusion model group(P0.05). TLR2 and TLR4 expression levels were significantly reduced in the SHP treatment and Astragaloside group as compared with the pseudo-operation group(P0.05).Further,the high-dose SHP group showed significantly less renal damage score and decreased levels of TLR expression than those of low-dose SHP group and Astragaloside group(all P0.05).Conclusion:SHP can alleviate the renal structural and functional damage caused by IRI-induced AKI in rats by reducing the damage of renal pathology,which may reduce inflammatory cytokine levels by downregulating the expression of TLRs in renal tissue in a dose-dependent manner.     

复方肾华片对急性脑缺血再灌注损伤大鼠肾小管Na+-K+-AT酶的调节作用

Objective:To observe the effect of Compound Shenhua Tablet(复方肾华片,SHT) on the sodiumpotassium-exchanging adenosinetriphosphatase(Na~+-K~+-ATPase) in the renal tubular epithelial cells of rats with acute ischemic reperfusion and to investigate the mechanisms underlying the effects of SHT on renal ischemic reperfusion injury(RIRI).Methods:Fifty male Wistar rats were randomly divided into the sham surgery group,model group,astragaloside group[150 mg/(kg-d)],SHT low-dose group[1.5 g/(kg·d)]and SHT high-dose group[3.0 g/(kg·d)],with 10 rats in each group.After 1 week of continuous intragastric drug administration,surgery was performed to establish the model.At either 24 or 72 h after the surgery,5 rats in each group were sacrificed,blood biochemistry,renal pathology,immunoblot and immunohistochemical examinations were performed,and double immunofluorescence staining was observed under a laser confocal microscope.Results:Compared with the sham surgery group,the serum creatinine(SCr) and blood urea nitrogen(BUN) levels were significantly increased,Na~+-K~+-ATPase protein level was decreased,and kidney injury molecule-1(KIM-1) protein level was increased in the model group after the surgery(P0.01 or P0.05).Compared with the model group,the SCr,BUN,pathological scores,Na~+-K~+-ATPase,and the KIM-1 protein level of the three treatment groups were significantly improved at 72 h after the surgery(P0.05 or P0.01).And the SCr,BUN of the SHT low- and high-dose groups,and the pathological scores of the SHT high-dose group were significantly lower than those of the astragaloside group(P0.05).The localizations of Na~+-K~+-ATPase and megalin of the model group were disrupted,with the distribution areas overlapping with each other and alternately arranged.The severity of the disruption was slightly milder in three treatment groups compared with that of the model group.The results of immunofluorescence staining showed that the SHT high-dose group had a superior effect as compared with the astragaloside group and the SHT low-dose group.Conclusions:The SHT effectively alleviated RIRI caused by ischemic reperfusion,promoted the recovery of the polarity of renal tubular epithelial cells,and protected the renal tubules.The therapeutic effects of SHT were superior to those of astragaloside as a single agent.     

Significance of Rosiglitazone Inhibiting TLR4 Expression in Partial Hepatic Ischemia/Reperfusion of Mice

The effect of rosiglitazone as the ligand of peroxisome proliferator-activated receptor γ (PPARγ) inhibiting the TLR4 expression in ischemic lobes in partial hepatic ischemia/reperfusion injury (IRI) in BABL/C mice and the action of rosiglitazone inhibiting the TLR4 receptor-mediated inherent immune response were investigated. The model of the mouse partial hepatic ische- mia/reperfusion injury was established. All the animals were randomly divided into 3 groups: rosiglitazone group, vehicle (dimethylsulphoxide, DMSO) group and sham operation group. The hepatic samples were collected when mice were sacrificed 0, 2, 4 and 6 h after reperfusion following 1 h ischemia to analyze the acute phase of hepatic IRI. The dynamic expression of TlR4 mRNA was de- tected quantitatively by real-time-PCR, and the levels of TNF-α, IL-10 and ALT in portal vein were determined in all groups. After restoration of blood supply, the expression of TLR4 mRNA in ischemic lobes was detected in 0, 2, 4 and 6 h after reperfusion following 1 h ischemia in rosiglitazone group and vehicle group. The most intensive expression of TLR4 mRNA was present at 4 h after reperfusion in ischemic lobes in vehicle group. As compared with vehicle group, the expression of TLR4 mRNA in ischemic lobes in rosiglitazone group was significantly decreased at 4 h after reper- fusion. The level of IL-10 in portal vein was markedly up-regulated in rosiglitazone group as compared with vehicle group. Contrarily, the levels of TNF-α and ALT in portal vein were markedly down-regulated in rosiglitazone group as compared with vehicle group at every time point in mouse partial hepatic IRI model. Rosiglitazone could alleviate the hepatic IRI by inhibiting TLR4 receptor-mediated inherent immune response.     

China introduce new bird flu measures ahead of spring period

Background Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury （IRI）. Toll-like receptor 4 （TLR4）, expressed on several liver cell types, and the nuclear factor-kappa B （NF-KB） signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-KB signaling pathway activation in a murine model of partial hepatic IRI. Methods Wild-type mice （WT, C3H/HeN） or TLR4 mutant mice （C3H/HeJ） were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-KB activation was determined by electrophoretic mobility shift assay （EMSA）. The levels of tumor necrosis factor-a （TNF-a） and intedeukin-1β （IL-1β） in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay （ELISA）. Results The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-KB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice （P〈0.01）. Endotoxin in each group was undetectable. The levels of TNF-α and IL-1β in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice （P〈0.01）. Conclusions The TLR4/NF-KB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-KB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.     

Haoqin Qingdan Decoction (蒿芩清胆汤) and Ribavirin Therapy Downregulate CD14 and Toll-Like Receptor 4 in Febrile Disease with Dampness-Heat Syndrome in A Mouse Model

Objective: To evaluate the effect of Chinese medicine Haoqin Qingdan Decoction(蒿芩清胆汤, HQD) for febrile disease dampness-heat syndrome(FDDHS). Methods: Forty mice were divided into four groups, including normal control, FDDHS(induced by Radix et Rhizoma Rhei recipe and influenza virus A1 FM1 model), HQD, and the ribavirin groups(10 in each). The normal control and FDDHS groups were administered normal saline. HQD and the ribavirin groups were administered HQD and ribavirin intragastrically once daily at a dose of 64 g/(kg·d) and 0.07 g/(kg·d), respectively for 7 days. Lethargy, rough hair, diarrhea, tongue color and sole color were evaluated for pathological changes in morphology. The tongue and lung tissues were collected for histology. The CD14 and toll-like receptor 4(TLR4) expression levels were measured using real-time quantitative polymerase chain reaction. Results: More than 80% of the FDDHS mice showed hypokinesia and lethargy, and pathological changes associated with rough hair, diarrhea, tongue color and sole color. With advanced treatment for 7 days, the thick greasy tongue fur of the HQD and ribavirin groups were thinner than that of the FDDHS group(P0.05), and it was the thinnest in the ribavirin group as compared with that in other groups(P0.05). The CD14 and TLR4 expression levels in the lung tissues of HQD and ribavirin groups significantly delined compared with the model group(P0.05 or P0.01). CD14 was down-regulated more remarkably in the HQD group compared with the ribavirin group(P0.05), whereas the converse was true with TLR4(P0.05). Conclusions: We established a FDDHS mouse model showing systemic clinical symptoms. Both HQD and ribavirin can inhibit the expression of CD14 and TLR4 in FDDHS mice, while the effect of ribavirin might be much more violent. The expression changes of CD14 and TLR4 consistently refers to lipopolysaccharide, the commonly and hotly inducing factor in FDDHS.     

The effect of Vaccinium uliginosum on rabbit retinal structure and light-induced function damage

Objective:To study the effect of Vaccinium uliginosum L,(VU) on the electroretinogram(ERG) and retinal pathological changes in rabbits after light-induced damage.Methods:Twenty-eight Chinchilla rabbits were randomly divided into four groups:administration beforehand(A),administration after injury(B),light injury without administration(C),and blank(D) groups.After a 4-week administration of VU homogenate at 4.8 g/(kg-d) once a day in group A,ERG in groups A,B and C were recorded according to the standards set by the International Society for Clinical Electrophysiology of Vision(ISCEV).Except for group D,the groups were then exposed to strong light.Just after that,group A stopped receiving VU treatment and group B started to receive it.Then ERGs in all groups were recorded after 1 day,1 week,and 2 weeks.Throughout the whole process groups which were not fed with VU were fed with normal saline.Finally,the tissues and structures of all the groups were observed and the thickness of the outer nuclear layers(ONL) was measured.Results:(1) After 4-week feeding with VU,the latency time of ERG in group A became shorter than those in the other groups and the amplitude increased.After being exposed to strong light,the latency time lengthened and amplitude decreased in all the injury groups,but comparing at each time point,the measured values in group A were better than those in group C.With the accumulation of VU,the ERG in group B improved,and finally,all of the detected values became better than those in group C.(2) Retinae in group D were normal in histology and the layers were in order but those in group C became disarranged.The injuries in groups A and B were minor compared with those in group C. The thickness of the ONL in group C was significantly thinner than in the other groups(all P=0.000),and that in groups A and B was thicker than that in group C,although thinner than in group D.That in group A was thicker than in group B.Conclusions:VU can relieve the injury to rabbit retinae exposed to normal day and night rhythm, alleviate the harm caused by light when used beforehand,and repair the light damage to the retina.     

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells (HPECs) to investigate their roles in innate immune response of the endocervix.Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence (IF). Cultured HPECs were divided into lipopolysaccharide (LPS) group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs.Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours (P <0.01), however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-α in a time-dependent manner (24 hours: P <0.05; 48 hours: P <0.01, compared with control group). Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line.Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.     

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells (HPECs) to investigate their roles in innate immune response of the endocervix.Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence (IF). Cultured HPECs were divided into lipopolysaccharide (LPS) group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs.Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours (P <0.01), however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-α in a time-dependent manner (24 hours: P <0.05; 48 hours: P <0.01, compared with control group). Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line.Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.     

Short-term inhalation of nitric oxide inhibits activations of toll-like receptor 2 and 4 in the lung after ischemia-reperfusion injury in mice

In order to investigate the effects of different terms of inhaled nitric oxide (NO) preconditioning with low concentration on the activations of Toll-like receptor 2 and 4 (TLR2/4) in the lung ischemia-reperfusion (IR) injury in mice, we divided the male C57BL mice into five groups: sham (S) group, IR group, NO 1-min preconditioning group (15 ppm NO inhalation for 1 min before ischemia, NO 1-min), NO 10-min preconditioning group (15 ppm NO inhalation for 10 min before ischemia, NO 10-min), NO 60-min preconditioning group (15 ppm NO inhalation for 60 min before ischemia, NO 60-min). The changes of partial pressure of oxygen in artery (PaO 2 ), left lung wet-to-dry weight ratio (W/D), and myeloperoxidase (MPO) in the injured lung were measured in every group at 6th h of reperfusion after 60 min of left lung ischemia. The changes of TLR2/4 activations and plasma TNF-α were measured in this procedure in additional mice. As compared with IR group, PaO 2 increased, MPO and W/D decreased evidently after reperfusion in NO 10-min group. The changes in NO 60-min group were similar to those in NO 10-min group. There was no difference between NO 1-min and IR group. In NO inhalation group, the expressions levels of TLR2/4 mRNA and proteins were diminished, TNF-α concentrations were decreased, and the lung injuries were ameliorated effectively. We concluded that short term inhalation of NO protected lung IR injury. But the protective effect of NO was not increased with extension of inhaled NO. Inhaled NO could inhibit the activations of TLR2/4 in the lung after IR injury. TLR signal pathway might contribute to the effect of protection with NO in this model.     

Effect of Soothing Gan (Liver) and Invigorating Pi (Spleen) Recipes on TLR4-p38 MAPK Pathway in Kupffer Cells of Non-alcoholic Steatohepatitis Rats

Objective: To investigate the mechanism of inflammatory-mediated toll-like receptor 4(TLR4)-p38 mitogen-activated protein kinase(p38 MAPK) pathway in Kupffer cells(KCs) of non-alcoholic steatohepatitis(NASH) rats and the intervention effect of soothing Gan(Liver) and invigorating Pi(Spleen) recipes on this pathway. Methods: After 1 week of acclimatization, 120 Sprague-Dawley male rats were randomly divided into 8 groups using a random number table(n=15 per group): normal group, model group, low-dose Chaihu Shugan Powder(柴胡疏肝散, CHSG) group(3.2 g/kg), high-dose CHSG group(9.6 g/kg), low-dose Shenling Baizhu Powder(参苓白术散, SLBZ) group(10 g/kg), high-dose SLBZ(30 g/kg) group, and low-and highdose integrated recipe(L-IR, H-IR) groups. All rats in the model and treatment groups were fed with a high-fat diet(HFD). The treatments were administrated by gastrogavage once daily and lasted for 26 weeks. The liver tissues were detected with hematoxylin-eosin(HE) and oil red O staining. Levels of liver lipids, serum lipids and transaminases were measured. KCs were isolated from the livers of rats to evaluate the mRNA expressions of TLR4 and p38 MAPK by real-time fluorescence quantitative polymerase chain reaction, and proteins expressions of TLR4, p-p38 MAPK and p38 MAPK by Western blot. Levels of inflammatory cytokines including tumor necrosis factor α(TNF-α), interleukin(IL)-1 and IL-6 in KCs were measured by enzyme-linked immunosorbent assay. Results: After 26 weeks of HFD feeding, HE and oil red O staining showed that the NASH model rats successfully reproduced typical pathogenesis and histopathological features. Compared with the normal group, the model group exhibited significant increases in body weight, liver weight, liver index, serum levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol, and aspartate aminotransferase as well as TC and TG levels in liver tissues, and significant decrease in serum level of high-density lipoprotein cholesterol(P0.05 or P0.01), while those indices were significantly ameliorated in the H-IR group(P0.05 or P0.01). Higher levels of TNF-α, IL-1 and IL-6 in KCs were observed in the model group compared with the normal group(P0.01). Significant decreases in TNF-α, IL-1 and IL-6 were observed in the H-SLBZ, H-IR and L-IR groups compared with the model group(P0.05 or P0.01). The m RNA expressions of TLR4 and p38 MAPK and protein expressions of TLR4, p38 MAPK and p-p38 MAPK in KCs in the model group were significantly higher than the normal group(P0.01), while those expression levels in the L-IR and H-IR groups were significantly lower than the model group(P0.05 or P0.01). Conclusions: Inflammation in KCs might play an important role in the pathogenesis of NASH in rats. The data demonstrated the importance of TLR4-p38 MAPK signaling pathway in KCs for the anti-inflammatory effect of soothing Gan and invigorating Pi recipes.     

Study on the Protective Effect of Shengmai San(生脉散) on the Myocardium in the Type 2 Diabetic Cardiomyopathy Model Rat

Objective:To study the effect of Shengmai San(生脉散Pulse-activating Powder) in protecting myocardium in the rat of the type 2 diabetic cardiomyopathy(DCM) model.Methods:The DCM rat model was established by combination of insulin resistance induced by a high-fat diet with intraperitoneal injection of high dose streptozotocin(50 mg/kg).And these rat models were randomly divided into three groups:a normal group(n=12,one of them died),a model group(n=15) and a Shengmai San group(treatment group,n=15).The damage of the myocardium was assessed by electrocardiogram at the twelfth week after modeling,and the blood glucose,cholesterol and triglyceride levels were determined;the content of the left cardiac ventricle myocardial collagen was quantified by Masson staining test;the level of myocardial cell apoptosis was detected with TUNEL apoptosis detection kit;the damage extent of the myocardial sub-cellular structures was observed by electron microscopy;the expression levels of cardiac TSP-1(Thrombospondin-1),TGF-β1(Transforming Growth Ffactor-β) and TRB-3(Tribbles homolog 3) proteins were detected by immunohistochemical method;the expression levels of cardiac TSP-1,A-TGF-β1 and L-TGF-β1 proteins were detected by Western blotting;and the expression levels of TSP-1 and TRB-3 mRNAs were detected by real-time quantitative PCR.Results:Compared with the control group,the blood glucose,cholesterol,triglycerides levels in both the model groups and the Shengmai San group were significantly decreased;the myocardial tissue was less damaged and the collagen content was reduced in the Shengmai San group;the myocardial sub-cellular structure was injured to a lesser extent;the expression levels of myocardial TSP-1,TGF-β1,TRB-3,and TSP-1,A-TGF-β1,L-TGF-β1 and chymase were decreased,and the expression levels of TSP-1 mRNA and TRB-3 mRNA were decreased in both the model groups and the Shengmai San group(the latter was better),.Conclusion:Shengmai San can inhibit myocardial fibrosis in the rat of diabetic cardiomyopathy,and significantly delay the formation of diabetic cardiomyopathy in hyperglycemia rats through multiple pathways.     

Effect of ω-3 polyunsaturated fatty acid on toll-like receptors in patients with severe multiple trauma

This study examined the effects of ω-3 polyunsaturated fatty acid(ω-3PUFA) on the expression of toll-like receptor 2(TLR2),toll-like receptor 4(TLR4) and some related inflammatory factors in peripheral blood mononuclear cells(PBMCs) of patients with early-stage severe multiple trauma.Thirty-two patients who were admitted to the Department of Traumatic Surgery,Tongji Hospital(Wuhan,China) between May 2010 and November 2010,and diagnosed as having severe multiple trauma with a injury severity score(ISS) no less than 16,were enrolled in the study and divided into two groups at random(n=16 in each):ω-3PUFA group and control group in which routine parenteral nutrition supplemented with ω-3PUFA or not was administered to the patients in two groups for consecutive 7 days.Peripheral blood from these patients was collected within 2 h of admission(day 0),and 1,3,5 and 7 days after the nutritional support.PBMCs were isolated and used for detection of the mRNA and protein expression of TLR2 and TLR4 by using real-time PCR and flow cytometry respectively,the levels of NF-κB by quantum dots-based immunofluorescence assay,the levels of TNF-α,IL-2,IL-6 and COX-2 by ELISA,respectively.The results showed that the mRNA and protein expression of TLR2 and TLR4 in PBMCs was significantly lower in ω-3PUFA group than in control group 5 and 7 days after nutrition support(both P<0.05).The levels of TNF-α,IL-2,IL-6 and COX-2 were found to be substantially decreased in PBMCs in ω-3PUFA group as compared with control group at 5th and 7th day(P<0.05 for all).It was concluded that ω-3PUFA can remarkably decrease the expression of TLR2,TLR4 and some related inflammatory factors in NF-κB signaling pathway in PBMCs of patients with severe multiple trauma,which suggests that ω-3PUFA may suppress the excessive inflammatory response meditated by the TLRs/NF-κB signaling pathway.     

加味竹叶石膏汤及其成分可预防大鼠放射性食管炎的发生

Objective:To investigate the effect of Modified Zhuye Shigao Decoction(加味竹叶石膏汤,MZSD)and its components on preventing radiation esophagitis of rats.Methods:One hundred Wistar rats were randomly divided into 5 groups,including the control group,radiation model group,MZSD group,Zhuye Shigeo Decoction(竹叶石膏汤,ZSD)group,and added Ingredients group,20 rats in each group.The model of radiation esophagitis of rat was established by once local radiation of 40 Gy(330 Mulmin)with a high energy linear accelerator.The administration of Chinese medicine was continued for 14 days from 7 days before radiation application in the three treatment groups.On the 7th and 14th day,the serum was isolated and the levels of inflammatory cytoldnes tumor necrosis factor(TNF-α),interleukin 1 13(IL-1β)and IL-8 were tested.The pathological slices of esophagus were obtained,and the pathological changes were observed.During the whole process,weight and food intake were recorded each day.Results:On the 7th day after radiation,the esophagus of rats in the MZSD group was almost intact,and the pathological injury score was significantly lower than that of the radiation model group,ZSD group and added ingredients group(P0.01).Compared with the control group,the body weight and food intake of rats in the radiation model group were significantly decreased,and the levels of TNF-α,IL-1β and IL-8 were significantly increased(P0.05 or P0.01),while the MZSD group showed a significant increase in body weight and food intake,and a significant decrease in the levels of TNF-α,IL-1β and IL-8 compared with the radiation model group,ZSD group and added ingredients group(P0.05 or P0.01).Conclusion:MZSD prevents the development of radiation esophagitis probably by inhibiting the generation and release of the inflammatory cytoldnes TNF-α,IL-1β and IL-8.     

通络方对糖尿病大鼠C型钠尿肽及钠尿肽B型受体表达的调节作用

Objective: To investigate the expression of C-type natriuretic peptides (CNP) and natriuretic peptide receptor-B (NPR-B) receptor in diabetic rats renal cortex, and the regulation by Tongluo Recipe (通络方, TLR). Methods: Sixty male SD rats were divided into 3 groups: the normal control group, diabetic model group and diabetic TLR group. Each group was further divided into two subgroups of ten in each, according to 4-week or 12-week observation period. Streptozotocin (STZ)-induced diabetic rats were treated with TLR (110 g?kg-1?d-1) for 4 and 12 weeks, respectively. (1) The essential information was collected for comparing renal mass, serum creatinine and 24 h urine albumen on each group was calculated. (2) CNP mRNA and NPR-B mRNA were detected by realtime-polymerase chain reaction (PCR) on rats renal cortex. (3) Concentration of CNP on renal cortex or serum were analyzed by enzyme-linked immunosorbent assay (ELISA). (4) Pathological evaluation and NPR-B immunostaining for renal tissue were also performed. Results: (1) CNP and NPR-B mRNA levels were detected in each treated or untreated group, with slight elevated in untreated diabetes rats administrated with STZ after 4-week and CNP mRNA level remarkable elevated at 39.21 times higher than normal control group after 12 weeks, but NPR-B mRNA level showed a remarkably down-regulation at 98.07% after 12 weeks. CNP mRNA of TLR-treated group was also elevated after 12-week treatment, but less than untreated group. (2) Concentrations of CNP in renal cortex were obviously increased in treated or untreated diabetes rats, within these groups the treatment of TLR was found more significantly on prompting CNP concentration. Comparing to normal group, serum concentrations of CNP were also increased in treated or untreated diabetic groups, but there was no difference between these diabetic groups. (3) Renal lesions like glomerular volume increased are observed mostly in the relative early stage after 4 weeks. Although TLR treated group had no significant difference in their glomerular volume, the degrees of injury of glomerulus were ameliorated, as well as the NPR-B immunostaining enhanced in glomerulus. Weakly positive immunostaining of NPR-B are observed in glomerulus of normal control, and negative in glomerulus of untreated diabetes rats administrated with STZ after 12 weeks, whereas TLR-treatment groups showed a little enhancement. Conclusion: CNP and NPR-B showed different characteristic on renal cortex at different pathological period in diabetes rats, and TLR regulated their expression.     

Effect of Compound Laser Acupuncture-Moxibustion on Blood Glucose, Fasting Insulin and Blood Lipids Levels in Type 2 Diabetic Rats

Objective: To investigate the effect of compound laser acupuncture-moxibustion on blood glucose, fasting insulin and blood lipids levels in type 2 diabetes mellitus(T2DM) rats. Methods: Forty male Wistar rats were randomly divided into 4 groups, including the normal group, model control group, laser group and sham laser group(n=10 per group). The rats in the normal group were fed with a standard diet. Rats in other groups were fed with a high-sugar and high-fat diet for 4 weeks, then intraperitoneally injected with 1% streptozotocin to induce T2 DM model. The laser group was irradiated by 10.6 μm and 650 nm compound laser on bilateral Pishu(BL 20), Shenshu(BL 23) and Sanyinjiao(SP 6) for 5 min, 6 times a week for 5 weeks. The sham laser group received the same treatment as the laser group, but without laser output. The model control group and normal group were not treated. Blood glucose levels were measured before and after 1, 2, 3, 4 and 5 weeks of treatment. The serum levels of fasting insulin, total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL), and high-density lipoprotein(HDL) were analyzed after the last treatment. Results: The blood glucose levels in the model control group increased during the 5 weeks of treatment compared with the normal group(P0.05), while those in the laser group were significantly lower than the model control group after weekly treatment(P0.01 or P0.05). After 1, 2 and 3 weeks of treatment, the blood glucose levels in the laser group decreased obviously compared with the sham laser group(P0.01 or P0.05). Compared with the normal group, the levels of fasting insulin, TC and LDL in the model control group notably increased(P0.01 or P0.05), while their levels in the laser group were significantly lower than the model control group after 5 weeks of treatment(P0.05 or P0.01). However, no statistically significant differences were observed in TG or HDL levels among the 4 groups(P0.05). Conclusion: The compound laser acupuncture-moxibustion of 10.6 μm and 650 nm had positive effects on the regulation of hyperglycemia and insulin resistance in T2 DM rats, which may be a potential treatment for T2 DM, and also provide an alternative to the traditional acupuncture and moxibustion therapy.     

Redox factor-1 may mediate the repair of multiple organ injuries after liver transplantation

AIM: To examine the relationship between multiple-organ injuries and expression of redox factor-1 (Ref-1) in the early period after liver transplantation. METHODS: A total of 150 adult male Wister rats were divided randomly into three groups: liver transplantation, sham surgery, and untreated control. After liver transplantation, animals were sacrificed at different time points. Hepatic and renal functions were analyzed by serology. Histology, apoptotic levels, and Ref-1 expression were examined by immunohistochemistry in the liver, kidneys, intestines, and lungs. RESULTS: Serum levels of alanine aminotransferase and aspartate aminotransferase peaked 6 h after liver transplantation and decreased appreciably after 12 h in the transplantation group, suggesting that the degree of liver injury in the early period after transplantation peaked at 6 h and then decreased. Pathological analyses showed that hepatic tissues were more severely injured in the transplantation group than in the sham and untreated groups. A considerable number of infiltrating inflammatory cells was observed around the portal vein in the transplantation group. Injuries to the kidneys, intestines, and lungs were milder after liver transplantation. Apoptotic levels increased after liver transplantation in all four organs examined. Ref-1 expression was higher in the transplantation group in the early period after liver transplantation than in the sham surgery and untreated control groups. CONCLUSION: Ref-1 expression induced by ischemia–reperfusion injury may have a critical role in repairing multiple-organ injuries after liver transplantation.     

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors （TLRs） and human defensins （HD） have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells （HPECs） to investigate their roles in innate immune response of the endocervix. Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence （IF）. Cultured HPECs were divided into lipopolysaccharide （LPS） group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs. Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours （P 〈0.01）, however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-a in a time-dependent manner （24 hours： P 〈0.05; 48 hours： P 〈0.01, compared with control group）. Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line. Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.