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Objective To observe the effects and mechanism of Guiqi Yiyuan Ointment on immune function in 12C6+ beam bystander-effect rats.Methods Totally 60 Wistar rats were randomly divided into blank group,model group,positive control group,and TCM low-,medium-,and high-dosage groups,with 10 rats in each group.TCM low-,medium-,and high-dosage groups were given 1.2,2.4,and 4.8 g/kg Guiqi Yiyuan Ointment for gavage,and the blank group,model group and positive control group were given equal dose of sodium chloride solution for gavage for 14 days.Positive control group received intraperitoneal injection of amifostine 30 minutes before modeling.On the 15th day,except the blank group,the other groups were given 4 Gy 12C6+ beam of single irradiation on the right lung.After irradiation,each group was given gavage for 3 days.The general condition of rats was observed,peripheral blood leukocyte(WBC),red blood cell(RBC),hemoglobin(Hb),platelet(PLT)levels were detected,ELISA was used to detect the contents of serum interleukin(IL)-4 and interferon-γ(IFN-γ),lung and spleen organ indices were calculated,HE staining was used to observe the pathological morphology of left lung and spleen tissues,and flow cytometry was used to detect the proportion of CD4+ and CD8+ T lymphocytes in the spleen,qPCR was used to detect the expressions of T-bet and GATA-3 mRNA in right lung and spleen tissues.Results Compared with the blank group,the model group rats showed a decrease in peripheral blood WBC,an increase in RBC,Hb,PLT,and an decrease in serum IL-4 and IFN-γ contents;the organ indices of left lung and spleen was significantly reduced,the alveoli were deformed,the alveolar walls became thicker,the alveolar cavities shrank,the lung tissue was significantly bleeding,the spleen tissue structure was severely damaged,the splenic sinus cavity became narrower,and the splenic cord showed fibrous hyperplasia;the proportion of CD4+ T lymphocytes and CD4+/CD8+ ratio in spleen tissue of the model group decreased,while the proportion of CD8+ T lymphocytes increased,the expression of T-bet mRNA in the left lung and spleen tissues decreased,while the expression of GATA-3 mRNA increased,with statistical significance(P<0.05).Compared with the model group,the positive control group and TCM medium-and high-dosage groups showed an increase in peripheral blood WBC,an decrease in RBC,Hb,PLT,an increase in serum IL-4 and IFN-γ contents;the index of left lung and spleen organs increased in TCM high-dosage group;the pathological damage of left lung and spleen tissues in each treatment group alleviated to varying degrees,while the proportion of CD4+ T lymphocytes and CD4+/CD8+ ratio in spleen tissue of the positive control group and TCM high-dosage group increased,the proportion of CD8+ T lymphocytes decreased,the expression of T-bet mRNA in left lung and spleen tissues increased,and the expression of GATA-3 mRNA decreased,with statistical significance(P<0.05).Conclusion Guiqi Yiyuan Ointment can alleviate the lung and spleen injury of rats induced by 12C6+ beam radiation.Its mechanism is related to regulating the proportion of CD4+,CD8+ T lymphocytes and the expression of T-bet and GATA-3,and recovering cellular immune function.
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ObjectiveTo validate the alleviating effect of Gegen Qinliantang （GGQLT） on insulin resistance in db/db diabetic mice by regulating the silent information regulator 1 （SIRT1）/forkhead transcription factor O1 （FoxO1） autophagy pathway. MethodSeventy-five SPF-grade spontaneous type 2 diabetic db/db mice and 15 control db/m mice were selected and maintained on regular feed for one week before measuring blood glucose. They were randomly divided into six groups， with 15 mice in each group. The groups included a normal group （physiological saline， 0.2 g·kg-1）， a metformin group （0.2 g·kg-1）， high-， medium-， and low-dose GGQLT groups （31.9， 19.1， 6.9 g·kg-1）， and a model group （physiological saline， 0.2 g·kg-1）. They were orally treated with corresponding drugs for eight weeks， once daily. Fasting blood glucose （FBG） was measured using a Roche glucometer. Serum levels of high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， triglyceride （TG）， and total cholesterol （TC） were measured using an automated biochemical analyzer. Fasting serum insulin （INS） levels were determined using enzyme-linked immunosorbent assay （ELISA）， and the homeostasis model assessment of insulin resistance （HOMA-IR） was calculated. Western blot was used to detect the expression of Beclin-1， microtubule-associated protein 1 light chain 3 （LC3）， and SIRT1/FoxO1 autophagy pathway-related proteins in liver tissues. Immunohistochemistry was performed to assess the expression of SIRT1， FoxO1， Beclin-1， and LC3B proteins in liver tissues. Transmission electron microscopy was used to observe the formation of autophagosomes in the liver. ResultCompared with the normal group， the model group showed significant increases in FBG， FINS， HOMA-IR， TC， TG， LDL-C， and HDL-C levels （P<0.01）， and significant increases in the expression of SIRT1， Beclin-1， LC3， and FoxO1 proteins in liver tissues （P<0.01）. Transmission electron microscopy revealed the highest number of autophagosomes in the model group. Compared with the model group， the metformin group and the low-， medium-， and high-dose GGQLT groups showed significant decreases in serum FBG， FINS， HOMA-IR， TC， TG， LDL-C， and HDL-C levels （P<0.05， P<0.01）， significant decreases in the expression of SIRT1， Beclin-1， LC3 （P<0.05， P<0.01）， and up-regulated FoxO1 protein （P<0.01）. Transmission electron microscopy showed a reduction in the degree of autophagy in the treatment groups. Compared with the metformin group， the medium- and high-dose GGQLT groups showed significant decreases in FBG， FINS， and TG levels （P<0.01）， significant decreases in the expression of SIRT1， Beclin-1， and LC3 in liver tissues （P<0.05， P<0.01）， and reduced FoxO1 protein （P<0.01）. The high-dose GGQLT group showed reduced HOMA-IR， TC， LDL-C， and HDL-C levels （P<0.05， P<0.01）. Transmission electron microscopy revealed a significant reduction in autophagosomes in the medium- and high-dose GGQLT groups. ConclusionGGQLT can significantly improve glucose and lipid metabolism disorders， alleviate insulin resistance in db/db mice， and prevent and treat type 2 diabetes by activating the SIRT1/FoxO1 autophagy pathway.
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ObjectiveTo explore the mechanism of Gegen Qinliantang （GQT） in improving ectopic lipid accumulation in the liver of db/db mice with type 2 diabetes mellitus （T2DM） by regulating the adenosine monophosphate-activated protein kinase （AMPK）-forkhead box O3a （FoxO3a） autophagy axis， to provide a scientific basis for clarifying the hypoglycemic mechanism of GQT and its clinical application. MethodSeventy-five spontaneous T2DM db/db mice and 15 normal db/m mice were selected and maintained on a regular diet for one week， followed by the measurement of blood glucose. They were then randomly divided into six groups， with 15 mice in each group， including normal group （0.2 g·kg-1 saline）， metformin group （0.2 g·kg-1）， high-， medium， and low-dose GQT group （31.9， 19.1， 6.9 g·kg-1）， and model group （0.2 g·kg-1 saline）. The mice were orally administered the corresponding drugs once daily for 12 weeks. Fasting blood glucose （FBG） and glycated hemoglobin （HbA1c） were detected. Fasting insulin （FINS） and free fatty acid （FFA） levels were measured by enzyme-linked immunosorbent assay （ELISA）. Pathological changes in liver tissues were observed by hematoxylin-eosin （HE） staining. The protein expression levels of phosphorylated （p）-AMPK， p-FoxO3a， and autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ） and p62 were detected using Western blot. Immunofluorescence was used to detect the expression of hypoxia-inducible factor-1α （HIF-1α） in liver tissues. Real-time polymerase chain reaction （Real-time PCR） was performed to detect the mRNA expression of AMPK， FoxO3a， and LC3 in liver tissues. ResultCompared with the normal group， the model group showed pathological changes in liver tissues， increased FBG， HbA1c， FINS， and FFA levels （P<0.01）， increased protein expression levels of p-AMPK， p62， and HIF-1α， decreased protein expression levels of p-FoxO3a and LC3Ⅱ in liver tissues （P<0.01）， decreased mRNA expression of AMPK， and increased expression of FoxO3a （P<0.01）. Compared with the model group， the treatment groups showed relieved liver tissue lesions and decreased FBG， HbA1c， FINS， and FFA levels （P<0.01）. The expression of p-AMPK， p62， and HIF-1α increased， while the expression of p-FoxO3a showed a dose-dependent decrease in the high-dose GQT group. The expression of LC3Ⅱ increased in the metformin group and the high-dose GQT group （P<0.01）. The mRNA expression of AMPK showed a dose-dependent increase， and the expression of FoxO3a showed a dose-dependent decrease in the treatment groups （P<0.01）. ConclusionGQT can improve ectopic lipid accumulation in the liver of T2DM db/db mice， which may be related to the regulation of the AMPK-FoxO3a autophagy axis.
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Objective Based on HMGB1/TLR4/NF-κB pathway,to investigate the effects of Shenqi Yiliu decoction combined with cisplatin on H22 liver cancer tumor mice and the effects of related immune indicators.Methods 50 SPF grade male KM mice,10 mice were taken as blank group by random number table method,and the other 40 mice were replicated as H22 hepatocellular carcinoma tumor-bearing mice model.After successful replication of the model,the model mice were randomly divided into model group,cisplatin group(2.5×10-3 g·kg-1),Shenqi Yiliu decoction TCM group(27.03 g·kg-1),and Shenqi Yiliu decoction TCM(27.03 g·kg-1)combined with cisplatin(2.5×10-3 g·kg-1),10 mice in each group were treated for 13 d.Determine tumor suppression rate,spleen index and thymus index;HE observes changes in oncology pathology;streaming cells detect the level of CD4+T,CD8+T cells in the spleen tissue;PT-PCR and WB method detect genes and protein expression related to HMGB1/TLR4/NF-κB signaling pathways in tumor tissues.Results ①Compared with the blank group,the mean body mass and mouse spleen index,thymus index,CD4+ T cell level and CD4+T/CD8+T value were significantly lower and CD8+T cell level was higher in the model group(P<0.05);②Compared with the model group,the mean tumor mass decreased(P<0.05),tumor volume decreased(P<0.05),and body mass increased(P<0.05)in each treatment group,and the spleen index,thymus index,CD4+T cell level and CD4+T/CD8+T ratio increased and CD8+T cell level decreased in both the Chinese medicine group and the combination group,and the treatment effect was significant in the Chinese medicine group(P<0.05),and HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression in tumor tissues of mice were reduced,and the effect was significant in the combined group(P<0.05).③Compared with the cisplatin group,HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression were reduced in the tumor tissues of mice in the combination group(P<0.05).④HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression in tumor tissues of mice in the combined group were reduced compared with those in the Chinese medicine group(P<0.05).Conclusion Shenqi Yiliu decoction combined with cisplatin can effectively inhibit tumor growth and improve related immune indexes in H22 hepatocellular carcinoma tumor-bearing mice,and the mechanism may be related to the inhibition of HMGB1/TLR4/NF-κB signaling pathway activation.
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Objective:To establish an in vitro culture system of small intestinal organoid in normal mice and perform functional identification, and to provide an in vitro research tool for material transport in the intestine under chronic kidney disease. Methods:The small intestinal crypts of C57BL/6J mice were isolated, extracted and cultured in an in vitro three dimension culture system. The formation of small intestinal organoid was observed with inverted microscope. The tissue structure of the small intestinal organoid was observed by hematoxylin and eosin staining. The cellular composition of the small intestinal organoid was identified by immunofluorescence. The expression of substance absorption-related transporters in the small intestinal organoid was detected by real time fluorescence quantitative PCR. Results:The small intestinal crypts were successfully extracted. The organoids of small intestine and different intestinal segments were successfully constructed. The cultured organoids had vigorous proliferation ability and maintained proliferation ability after passing through generations. Immunofluorescence results showed that the small intestinal organoids expressed mucin2, chromogranin A, oflm4 and lysozyme, which were different types of intestinal cell biomarkers. The PCR results showed that small intestinal organoids expressed calcium, phosphate and sodium absorption-related transporters, and the mRNA expression levels of major transporters for sodium and phosphate absorption in different intestinal segments-like organs were consistent with those in vivo, which was consistent with the characteristics of small intestinal segmental absorption. Conclusions:The successful construction of small intestine and different intestinal segments organoids, and the first observation of the expression of substance absorption-related transporters in such organoid, provide a stable and convenient in vitro research tool for the development of intestinal substance transport in chronic kidney disease.
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Objective To investigate the mechanism of Guiqiyiyuan Ointment (GO) for preventing and treating 12C6+ beam radiation induced lung and kidney bystander effect to provide a new strategy for prevention and treatment of clinical radiation injury. Methods Sixty healthy male Wistar rats were randomly and equally divided into 3 groups: NC group, SR group (simple radiation 2ml/kg), GO group(GO 2ml/kg intragastric administration for 7 days). The right side of the lung was modeled by 12C6+beam radiation. After modeling, the rats were killed at 48h. The left lung, left and right kidney tissues were taken from the rats. The DNA methylation rate was detected by ELISA assay, pathological changes were observed by HE staining, and the expressions of Dnmt1, Dnmt3a and Dnmt3b were detected by immunohistochemistry. Results Compared with the NC group, the level of DNA methylation was decreased significantly (P<0.01), the left lung showed inflammation, no abnormal finding was seen in the left and right kidneys, and the expressions of Dnmt1, Dnmt3a and Dnmt3b were significantly increased in the SR group (P<0.01). Compared with the SR group, the level of DNA methylation was increased significantly (P<0.01), the left lung inflammation became better, and the expressions of Dnmt1, Dnmt3a and Dnmt3b were significantly decreased in the GO group (P<0.01). Dnmt1, Dnmt3a and Dnmt3b proteins were expressed in the cytoplasms of bronchial and renal tubular epithelial cells in all the groups. The NC group presented as light brown-brown staining, showing a weak positive expression, the SR group as brown-brown staining, showing astrong positive expression, and the GO group as light brown-brown staining, showing a moderate positive expression. Conclusion The GO can reduce the bystander effect caused by 12C6+ beam radiation, and its mechanism is related to improving the level of DNA methylation.
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Objective To observe the effects of Huangqi injection associated with a combined treatment on changes of hemodynamics indexes and brain natriuretic peptide (BNP) after cardiopulmonary resuscitation (CPR) in rabbit models with cardiac arrest (CA) to elucidate the mechanism of such cardiac protective effects. Methods Twenty-four New Zealand rabbits were randomly divided into four groups(each,n=6). The CPR model in rabbits was reproduced by using extra-corporal electric shock method(put through 50 V alternating current to quiver on chest). The animals in operation control group were given anesthesia,all kinds of catheter inserted into the body and tracheotomy,but no induction of ventricular fibrillation. In the epinephrine group,epinephrine (30 μg/kg)was injected into the right jugular vein by a catheter during CPR. In the sub-hypothermia group, epinephrine(30μg/kg)was injected into the right jugular vein via a catheter and 0.9%sodium chloride under 4℃at a rate of 1.0 mL · kg-1 · min-1 was pumped into the ear marginal vein simultaneously with the chest external compressions during CPR and the cooling of body surface sustaining at the target temperature (32-34℃) for 4 hours until the end of the experiment. In the combined treatment group,Huangqi injection(4 g/kg)was injected into the right jugular vein on the basis of treatment of sub-hypothermia group. Hemodynamics indexes such as left ventricular end-diastolic pressure(LVEDP),the maximal rate of left ventricular pressure increase/decline(±dp/dt max),and the mean arterial pressure(MAP)were dynamically monitored at 15 minutes before inducing ventricular fibrillation and at the early stage of post resuscitation for 30,60,120,180,240 minutes,and in the mean time the concentration of BNP was dynamically monitored at 15 minutes before inducing ventricular fibrillation and at the early stage of post resuscitation for 30,180,240 minutes. Results Compared with those of the operation control group,the levels of LVEDP and BNP of epinephrine,sub-hypothermia and combined treatment groups were gradually increased obviously,while ±dp/dt max and MAP were decreased significantly after successful CPR. The levels of LVEDP and BNP at any time point in the combined treatment group were markedly less than those of the epinephrine and sub-hypothermia groups,and along with the time extension,the level of LVEDP had a tendency of elevation,and the level of BNP had a tendency of gradual decrease,reaching peak and valley values respectively at 240 minutes after CPR〔LVEDP (mmHg,1 mmHg=0.133 kPa):6.56±0.21 vs. 12.57±0.33,9.54±0.24,BNP(ng/L):199±19 vs. 286±14, 251±29,all P<0.01〕,at each time point,the ±dp/dt max and MAP levels were significantly higher than those in epinephrine and sub-hypothermia groups,and they were gradually decreased with the time prolongation,reaching valley values at 240 minutes after CPR〔+dp/dt max(mmHg/ms):4 229±353 vs. 2 055±311,3 224±158,-dp/dt max(mmHg/ms):3 587±168 vs. 1 315±189,2 357±245,MAP(mmHg):82.02±1.81 vs. 44.15±1.17, 56.79±1.60,all P<0.01〕. Conclusion Huangqi injection associated with a combined treatment has obvious protective effects on heart after CPR in rabbits,and the mechanism may be related to improvement of hemodynamics indexes and inhibition of excessive expression of BNP.