Study On The Therapeutic Effect Of Acupoint Injection Combined With Traditional Chinese Medicine Prescription On H1N1 Induced Pneumonia In Mice

In recent years,a variety of highly pathogenic respiratory virus caused by viral pneumonia(human and livestock)have been outbreak or epidemic in local areas or worldwide.This kind of disease is mainly treated with supportive therapy and symptomatic treatment in clinic,but it’s lack of effective antiviral treatment.Acupuncture、moxibustion and Traditional Chinese medicine are important components of traditional Chinese(veterinary)medicine.This study created a mouse model of pneumonia induced by H1N1(Pr8)to explore the therapeutic effect and potential mechanism of acupoint injection combined with Chinese medicine prescription in order to provide new ideas and theoretical basis for the prevention and treatment of viral pneumonia.Methods:In order to create the mode,ICR mice(male,20±1g)were used to lie supine after anesthesia and 50 μL virus solution(104.4 EID50/50μL)was dripped into the nosel.On the second day of modeling,mice were treated once a day for 7 days.Treatment methods of each treatment group:The acupoint injection group injected 40 μL of 0.1%Vc+VBCo at Quchi,Zusanli and Tiantu points respectively;The non-target acupoint group injected 40 μL of 0.1%VC+VBCo on the left side of Tiantu and Quchi and Zusanli acupoint(0.5 cm parallel to the outside of each acupoint);The Chinese medicine group employed 120 μL of the corresponding Chinese medicine decoction(1 g/mL)by intragastric administration;The acupoint injection combined with traditional Chinese medicine group employed 120 μL of traditional Chinese medicine decoction(1 g/mL)by intragastric administration and then 0.1%Vc+VBCo 40 μL was injected into Quchi,Zusanli and Tiantu points respectively;The ribavirin group injected 120 μL of physiological saline solution containing 0.6 mg ribavirin by intraperitoneally.The clinical symptoms of each group were observed daily and the weight,temperature changes and the deaths of each group were recorded.On the 8th day of model establishment,the lung tissue samples of mice were collected under anesthesia.Using HE staining to observe lung tissue structure,RT-qPCR to detect the viral load,kit to detect changes in inflammatory factors and redox indicators,and Western Blot to detect the changes of NF-κB signaling pathway key protein expression in lung tissue.Results:(1)Clinical manifestations:On the first day of challenge,no obvious abnormality was observed in each group of mice;On the second day,the tongue of mice became fatter and bigger,the surface changed from smooth to dry,and the color changed from pink to dark purple;On the third day,the mice showed anorexia,back hair antithesis,agglomeration and decreased activity,at the same time,the feed intake and water intake of mice decreased;On the third day to the fifth day,feces became sticky(with hair on the surface),and on the sixth day to the seventh day,feces became small and hard;On the fourth day,the body weight of mice decreased significantly(P<0.05);On the fifth day,the body temperature of mice decreased significantly(P<0.01)and deaths appeared and the mortality of mice in D5,D6 and D7 groups was 10%,20%and 60%,respectively.Lung tissue lesion:On the second day of challenge,the contents of pro-inflammatory factors,the activity of oxidoreductase and the products of oxidation in lung tissue were significantly increased(P<0.05);On the third day,viral load in lung tissue increased significantly(P<0.001);On the fifth day,lung index of mice increased significantly(P<0.01);Inflammatory cells and red blood cells were infiltrated in the lung tissue,part of alveolar structures disappeared and alveolar septa thickened.(2)Acupoint injection can significantly reduce the mortality of mice and significantly prolong the survival time(P<0.05).Acupoint injection can significantly reduce the infiltration of inflammatory cells and red blood cells in the lung tissues of surviving mice;Significantly inhibit the expression of pro-inflammatory factors(TNF-α,IL-1β)(P<0.05),promote the expression of anti-inflammatory factors IL-10,significantly increase the activity of antioxidant enzymes(SOD,GSH-Px)(P<0.05),reduce the production of oxidation products MDA(P<0.05),thereby protecting the integrity of alveolar structure.Acupoint injection has no significant inhibitory effect on virus replication in lung tissues of surviving mice.(3)Compared with AI+DN-DC-2 group,the mortality of mice in AI+DN-DC-2 group was significantly decreased(P<0.05).Compared with AI group,the average survival time was significantly prolonged(P<0.05),and the viral load in lung tissue was significantly decreased(P<0.05).Compared with AI+DN-DC-1 group,the mortality of mice in AI+DN-DC-2 group was significantly reduced and the average survival time was significantly prolonged(P<0.05).There were no significant differences in lung index,inflammatory factors and oxidative stress indexes among the treatment groups.The combination of acupuncture and medicine significantly reduced inflammatory cell infiltration in lung tissue,inhibited NF-kB signaling pathway,weakened inflammation and oxidative stress response,and protected lung tissue structure.Conclusion:The model syndrome of pneumonia induced by H1N1(Pr8)strain in mice changed from the early stage(2-4 d)of Pi Fei Yang Xu and Zhuo Shi Nei Ting syndrome to the later stage(5-7 d)of Pi Shen Yang Xu Jia Yu syndrome,to the syndrome of Yin Sheng Nei Han,Si Zhi Jue Ni syndrome;Vc combined with VBCo acupoint injection can reduce lung inflammation and oxidative stress without affecting virus replication,and is superior to ribavirin in reducing 7-day mortality and prolonging average survival time of mice.The self-developmented Chinese herbal formula could significantly inhibit virus replication,reduce lung inflammation and oxidative stress injury,and was superior to Qingfeihuidu Decoction in reducing the 7-day mortality and prolonging the average survival time of mice.Acupoint injection and Chinese medicine prescription had synergistic protective effect on the model.