Therapeutic Effect Of Atomized Inhalation Of Tetrandrine Loaded Zinc-Alginate Nanogels On Bleomycin-Induced Interstitial Pneumonia In Rats

Lung cancer is the most common malignant tumor in the world.At present,epidermal growth factor receptor tyrosine kinase inhibitors are commonly used in clinical treatment,but these drugs can produce serious side effects,such as interstitial pneumonia.At present,the main strategy of interstitial pneumonia in the clinical treatment is to reduce the inflammatory response and delay the progress of pulmonary fibrosis.Glucocorticoids and immunosuppressants are the main treatment drugs,but the treatment method is relatively single,and there is no individualized treatment for the etiology and combined diseases.Since the glucocorticoids inhibit the immune system,these drugs are highly toxic and can lead to long-term dependence,and the chances and severity of infection may be increased.Therefore,in this study,tetrandrine,which has anti-inflammatory,anti-fibrosis,anti apoptosis,anti-tumor and other pharmacological effects,was selected as the candidate drug.The tetrandrine loaded zinc-alginate nanogels were prepared by reverse microemulsion method,which improved the disadvantages of low solubility and excessive pulmonary elimination,with ultrasonic atomized inhalation as administration to treat bleutomycin-induced interstitial pneumonia rats.In this study,the reaction mechanism and gelation mechanism of sodium alginate and Zn²⁺were analyzed by circular dichroism.Tetrandrine loaded zinc-alginate nanogels were prepared by reverse microemulsion method,the concentration of sodium alginate,the concentration of zinc chloride,the ratio of Span-80 and Tween-80 as the factors of investigation,nanogel particle size,polydispersity index（PDI）,Zeta potential as the response values,the preparation technology of tetrandrine loaded zinc-alginate nanogels was optimized by star design test.The characterization of tetrandrine loaded zinc-alginate nanogels were evaluated by particle size and potential analysis,transmission electron microscopy（TEM）,fourier transform infrared spectroscopy（FT-IR）and differential scanning calorimetry（DSC）.The drug loading rate,encapsulation rate and cumulative release rate of tetrandrine loaded zinc-alginate nanogels were determined by high performance liquid chromatography（HPLC）,and the release behavior of nanogels in vitro was evaluated.The antioxidant activity of nanogels in vitro was studied by 1,1-diphenyl-2-trinitrophenylhydrazine（DPPH）radical scavenging assay.The rat model of interstitial pneumonia was established by one-time tracheal infusion of bleomycin.The pulmonary delivery of tetrandrine loaded zinc-alginate nanogels was achieved by ultrasonic atomization.The therapeutic effects of tetrandrine loaded zinc-alginate nanogels at different concentrations on acute and chronic interstitial pneumonia were investigated.Micro-CT was used to scan and three-dimensional reconstruct lung tissue of rat,and the imaging characteristics of lung tissue were observed.H&E staining and Masson staining were used to observe the degree of inflammation and fibrosis in lung tissue of rats.The mechanism of tetrandrine loaded zinc-alginate nanogels on acute interstitial pneumonia and chronic interstitial pneumonia in rats was investigated by Real-time RT-PCR.The TUNEL staining assay was used to detect the number of apoptosis cell in the rats with acute and chronic stages.Immunofluorescence and immunohistochemical assay were used to detect the expression of fibrosis related proteins in the rats with chronic stages of interstitial pneumonia.The results of the central test showed that the concentration of sodium alginate was 1%,the concentration of zinc chloride was 0.25%,and Tween-80:Span-80 was 1:1.85,which were the optimal prescription of tetrandrine loaded zinc-alginate nanogels.Under TEM microscope,it can be observed that the nanogels are round in shape,smooth in surface,uniform in internal density and the particles size is less than 100 nm.At low magnification,it was found that the nanogel particles did not aggregate and had good dispersion.FT-IR,DSC results appearanced that tetrandrine was completely coated in tetrandrine loaded zinc-alginate nanogels system.The average loading rate and encapsulation rate of tetrandrine loaded zinc-alginate nanogels determined by HPLC were（1.59±0.07）%and（98.13±0.10）%.The results of in vitro release showed that the cumulative release rate of tetrandrine loaded zinc-alginate nanogels was 60.78%within 72 hours.The results of DPPH experiment appearanced that the antioxidant activity of tetrandrine loaded zinc-alginate nanogels was stronger than tetrandrine at the same concentration.Micro-CT images showed that large patches of infiltrating shadow appeared in the lung tissue of the model group on day 7,and obvious cable-like shadow appeared on day28.This indicated that there was obvious inflammatory damage in the lungs on day 7,and there were obvious signs of fibrosis in the lungs on day 28.In Masson staining and H&E staining sections of lung tissue,inflammatory cell infiltration was found in the model group on day 7,and inflammatory infiltration accompanied with collagen deposition on day 28.These results showed that acute inflammatory reaction mainly occurred in the lung tissue on day 7,and the lung tissue had changed from acute inflammatory phase to chronic inflammatory phase on day28,with significant fibrosis.The results of TUNEL staining appearanced that tetrandrine loaded zinc-alginate nanogels could inhibit the apoptosis of lung tissue cells in both acute and chronic stages.The results of Real-time RT-PCR,immunofluorescence,and immunohistochemical showed that the tetrandrine loaded zinc-alginate nanogels could treat interstitial pneumonia by inhibiting the inflammatory response（TNF-α,MCP-1）,apoptosis（Bax,Bcl-2,Bcl-x L）and slowing down fibrosis（TGF-β₁,α-SMA,Col-I,Col-III,CTGF,VEGF,β-catenin,FN,MMP-9,TIMP-1）.The results showed that ultrasonic atomization inhalation of tetrandrine loaded zinc-alginate nanogels had a therapeutic effect on interstitial pneumonia in rats,and provided a new therapeutic strategy for interstitial pneumonia.