Effect Of Bushen Gubiao Granules On Immunocompromised Mice And Its Safety Evaluation

Objevtive: Bushen Gubiao Granules is the classic prescription of Xu’s pediatrics for treatment of repeated respiratory tract infections.The main pathogenesis of repeated respiratory tract infections is immunodeficiency and disorder.Gavage administration for 4weeks,with Yupingfeng granules as the control group to study the regulatory effect of Bushen Gubiao Granules on the immune function of immunocompromised mice and evaluate the safety of Bushen Gubiao Granules by the maximum dose.It provides theoretical and experimental basis for the safety of Bushen Gubiao Granules in the prevention and treatment of repeated respiratory infections and the safety of clinical medication.Method: Experiment 1: 60 clean-grade ICR male mice were randomly divided into 6groups,that is,blank control group,model group,Yupingfeng granule group,low-dose group of Bushen Gubiao granules,middle-dose group of Bushen Gubiao granules,and Bushen Gubiao high-dose group,10 in each group.Except the blank group,the other groups were given intraperitoneal injection of cyclophosphamide at 50 mg / kg/ d for 3 consecutive days to establish a mouse model of immunodeficiency.After the model was successfully established,each group was given the corresponding medicine by gavage.The low,medium and high dose groups of Bushen Gubiao granule were given by gavage at 4.5g / kg / d,9g / kg / d and 18 g /kg / d respectively;the positive control group Yupingfeng granules were administered at2.25 g / kg / d,and the model group and the blank control group were given normal saline with an intragastric volume of 0.5mL / time,twice a day for 4 consecutive weeks.After the last dose,remove the feed and drink water freely.After the mice were anesthetized the next day,collecting blood by removing the eyeballs,then standing at room temperature for 2 hours,the blood was placed in a centrifuge and centrifuged at 2500 r / min for 15 minutes.The supernatant was sucked and filled into 0.3mL centrifuge tubes at-80 ℃ Store in the refrigerator for detecting immune indexes such as TNF-α,IL-2 and IFN-γ levels later.The mice were dissected,the spleens of the mice were harvested and weighed with an electronic balance,and the spleen coefficients of the mice in each group were calculated after the experiment.Subsequently,the splenic lymphocytes of the mice were extracted to detect the proliferation ability of splenic lymphocytes and the killing activity of NK cells.Experiment 2: 40 clean-grade ICR mice were selected,weighing 18-22 g,male and female,divided randomly into 2 groups according to body weight,namely,the blank group and the drug-administered group,with 20 in each group.The administration group was given the maximum concentration of Bushen Gubiao granules(1 mL containing 1.71 g of crudedrug)and the maximum gavage volume(0.4 ml / 10 g)twice a day at 6-hour intervals.The control group was given an equal volume of normal saline.Observe for 14 consecutive days,observe and record animal abnormalities(mental state,activity,fur condition,fecal secretion and other secretions),weight changes and death,etc.Grossly dissect the dead mice,and the rest of the mice will end the observation period.After gross anatomical observation,pathological tissue examination was performed from time to time.Results: 1.Changes in body weight of mice: Compared with the blank control group,the weight of the cyclophosphamide model mice lost weight,the difference was statistically significant(P <0.05);compared with the model group,after intragastric administration,the Yupingfeng granule group,Bushen Gujiao The body weight of mice in each dose group of the granules increased(P <0.05),the difference was statistically significant;there was no statistically significant difference between the low,medium and high dose groups of Bushen Gubiao Granules(P> 0.05).2.Spleen coefficient: Compared with the blank control group,the spleen coefficient of cyclophosphamide model mice decreased;compared with the model group,after oral administration,Yupingfeng granule group,Bushen Gubiao granule The spleen coefficient of mice in each dose group increased(P <0.05),the difference was statistically significant;the dose-effect relationship between the low,medium and high dose groups of Bushen Gubiao Granule was not statistically significant(P> 0.05).3.Spleen lymphocyte proliferation ability: Compared with the blank control group,the proliferation ability of spleen lymphocytes of mice in the cyclophosphamide model group was significantly decreased(P <0.01).Compared with the mice in the model group,the proliferative capacity of spleen lymphocytes of the various dose groups of Bushen Gubiao granule and Yupingfeng granule group after oral administration was increased,and was close to normal levels(P <0.05).4.Murine NK cell killing activity: Compared with the blank control group,the killing activity of NK cells in the model group was reduced(P <0.05).Compared with the model group,the NK cell killing activity of the Bushen Gubiao granule group and the Yupingfeng granule group were increased after intragastric administration,and the differences were statistically significant(P <0.05).The killing rate of Bushen Gubiao granules in the high-dose group was stronger than that in the low-dose group and Yupingfeng granule group(P <0.05).5.Changes of serum IL-2,IFN-γ and TNF-α levels: Compared with the blank control group,the serum IL-2 and IFN-γ levels in the model group decreased and the TNF-α levelincreased.The differences were statistically significant(P <0.05).The levels of IL-2 and IFN-γ in the group and Yupingfeng granule group increased,while the levels of TNF-αdecreased.The difference was statistically significant(P <0.05).The increase of IL-2 level in the middle and high dose group of Bushen Gubiao granule was better than that of Yupingfeng granule group(P <0.05).The middle and high dose groups of Bushen Gubiao Granule increased IFN-γ level better than the low dose group(P<0.05).There was no statistically significant difference in dose-effect relationship between TNF-α levels between different dosage groups of Bushen Gubiao Granules(P>0.05).6.A few minutes after administration,the mice in the acute toxicity experimental group had slightly reduced activity and returned to normal after 4 hours.On the second day of administration,the food intake of the mice decreased significantly,and then increased immediately,with no other abnormalities.During the observation period,the body weight of mice generally showed an increasing trend.Statistical analysis indicated that the difference was not statistically significant(P>0.05).During the observation period,the mice’s hair color was bright,and there were no reactions such as excitement,restlessness,vertical hair,raised eyes,writhing,etc;there were no obvious abnormalities in feeding,drinking,urination,etc.No mice died.After 14 days of observation,the mice were dissected,and the heart,liver,spleen,lung,kidney,stomach,intestine and other organs were visually observed.No obvious pathological changes such as bleeding,congestion,and edema were observed.Conclusions: 1.Intraperitoneal injection of cyclophosphamide can establish a mouse model of immunocompromised mice.After modeling,the mice have reduced diet,hug groups,reduced body mass,decreased spleen index,reduced spleen lymphocyte proliferation capacity and reduced NK cell killing activity.Modeling with cyclophosphamide was successful.2.Bushen Gubiao granules can obviously enhance the proliferation ability of spleen lymphocytes,NK cell killing activity of immunocompromised mice,and improve the immunity of the body.3.Bushen Gubiao granules can increase serum IL-2 and IFN-γ levels,reduce TNF-αlevels in immunocompromised mice,and enhance immune function by regulating cytokine levels.4.The commonly used dose of Bushen Gubiao granules is safe for oral administration.