Preparation And Characteristics Of Carboxymethyl Chitosan From Silkworm Pupa And Its Postoperative Anti-adhesion Ability In A Rat Cecal Abrasion Model

In this paper, we used waste silkworm pupae after silk reeling as experimental material. The silkworm pupa chitosan were obtained after pupa powder was dealt with the slow freezing-thawing at low temperature cycle. And then the silkworm pupa chitosan was modified into carboxylmethyl chitosan by chloroacetic acid. Its effects on fibroblast cells and postoperative intestinal adhesion in rats were studied in the paper.The silkworm pupa were dried in the oven at 80-100°C and then crushed into powder. The defatted pupa powder and pupa oil were obtained after the pupa powder was dealt with hexane. Then, silkworm pupa chitin was isolated from the defatted pupa powder by treatment with 7% Na OH to completely remove proteins, 5% HCl to remove inorganic salts, and finally with 30% H2O2 for decolouration. The chitin powder was treated with temperature cycling（-20 °C and 40°C） to obtain silkworm pupa chitosan. And then it was modified into carboxylmethyl chitosan by chloroacetic acid. The results showed that the DDA of the silkworm pupa chitosan was only 49.87 ± 0.86% and the viscosity-average molecular weight was 2.50×104. Although the DDA was low, its solubility in 2% HAc also could reach 96.73%. The substitution degree of silkworm pupa carboxymethyl chitosan was 1.63, it showed that the carboxymethyl chitosan we prepared was N,O-carboxymethyl chitosan which was belong to high substitution degree of carboxymethyl chitosan. The physical characteristics of silkworm pupa samples were analysised by SEM, XRD, DSC, FTIR, XPS and so on. The results of SEM showed that silkworm pupa chitosan and carboxymethyl chitosan samples were more uneven and fracture compared with standards. XRD, DSC and FTIR analysis results showed that the main molecular chain structures of silkworm pupa samples and reference standards had no substantial difference. But the crystallinity and thermal decomposition temperature of the silkworm pupa samples were lower compared with the standards.The influence of silkworm pupa carboxymethyl chitosan on L929 fibroblast proliferation was studied in this paper. The results showed that it could inhibit cell proliferation and reduce the gene expression of TGF-β₁ and VEGF. A rat cecal abrasion model was created as the cecum damage. The physiological and biochemical indexes and the changes of serum inflammatory markers were detected. Sixty male rats（200-250g） were randomly divided into three groups as follows: 0.9% normal saline solution（Group A）, medical anti-adhesion gel（Group B） and silkworm pupa carboxylmethyl chitosan anti-adhesion solution（Group C）. Two and three weeks after surgery, the animals were sacrificed and adhesion formation and inflammatory factors were evaluated. The results of the abdominal adhesion grade at the different time points showed that the mean adhesion score of Groups B and C were lower than Group A. The results of haematology parameters was indicated that the WBC counts in Groups B and C showed a significant decrease compared to Group A. The results of the inflammatory factors showed that the silkworm pupa carboxylmethyl chitosan significantly decreased the levels of TNF-α, IL-1β, IL-2, IL-6, IL-8. But there were no significant differences in IL-4 levels. In addition, the hydroxyproline levels showed that the silkworm pupa carboxylmethyl chitosan remarkably reduced the collagen level after surgery. The results of the histological analysis indicated that the silkworm pupa carboxylmethyl chitosan significantly decreased collagen synthesis, thereby preventing postoperative adhesion. The results of the expression of TGF-β₁ were showed that the silkworm pupa carboxylmethyl chitosan also reduced the expression of TGF-β₁, thereby reducing adhesion.In conclusion, this paper used the temperature cycling method to obtain the silkworm pupa chitosan and then it was modified into carboxylmethyl chitosan by chloroacetic acid. The results of cells culture showed that it could inhibit cell proliferation and reduce the gene expression of TGF-β₁ and VEGF. The results of in vivo tests showed that it could effectively prevent the occurrence of postoperative intestinal adhesion by raising the total antioxidant capacity, reducing the inflammatory factors in the cytokines and fibrous tissue hyperplasia. All of this laid a good foundation of the further development and utilization of silkworm pupa and also provided effective references for the development of more effective and affordable anti-adhesion drugs.