| Since ancient times,Chinese medicine has recorded that boiling silk cocoons with water was used to treat"xiao ke sickness",that is,diabetes.In recent years,studies have determined that sericin（SS）plays a role in the treatment of type 2 diabetes（T2DM）.Sericin can promote islet signal transduction,islet cell proliferation,and insulin secretion;it can also promote hepatic glycogen production,improve glucose tolerance and insulin resistance;at the same time,it has the effects of improving antioxidant capacity,reducing inflammatory response,liver and kidney damage,etc.thereby lowering blood sugar levels.Therefore,it is inferred that sericin enters the blood to participate in the systemic circulation after oral administration.As a natural protein,sericin needs to resist the digestion of gastrointestinal proteases and cross the gastrointestinal barrier after oral administration.At present,how sericin passes through the gastrointestinal barrier and whether it enters the systemic circulation and exerts a therapeutic effect on type 2 diabetes is still unclear.In response to this problem,this topic carried out research through in vitro simulated gastrointestinal digestion reaction experiments,oral/intraperitoneal injection animal experiments,and in vitro cell simulated intestinal epithelial cell layer absorption and transport experiments.The corresponding research results are as follows:Part 1:After oral administration of simulated sericin in vitro,the molecular weight of sericin becomes smaller,but it has anti-digestion properties after digestion and enzymatic hydrolysis in the gastrointestinal tract.Two methods were used for sericin extraction in this subject.（1）sericin was prepared by high temperature water extraction.After freeze-drying,sericin was white flakes,and its molecular weight was mainly distributed between25k～260k Da.After three hours of pepsin digestion in simulated gastric juice,there was no significant change in the molecular weight distribution range,but then after trypsin digestion in simulated intestinal juice,the molecular weight range became 25k～40k Da.（2）The molecular weight of sericin extracted from sodium carbonate is mainly concentrated between 10k and 25k Da,and it is a yellow flocculent powder after freeze-drying.After 3hours of digestion reaction in simulated gastric juice,the molecular weight distribution range did not change significantly.After 3 hours of simulated intestinal juice digestion reaction,the main molecular weight distribution range became between 10k～15k Da.The sericin extracted by the two methods has good anti-digestion properties,and the molecular weight of sericin extracted by the alkaline method is significantly lower than that of high temperature water extraction before and after enzymatic hydrolysis.Part Ⅱ:After gavage of sericin（SS-po）,intraperitoneal injection of sericin（SS-ip）and intraperitoneal injection of sericin after digestion in simulated gastrointestinal fluid（ad SS-ip）,all Sericin can be detected in the blood.It shows that sericin can enter the blood by oral or intraperitoneal injection,and participate in the systemic circulation,and the injection effect is better than that of oral administration.In order to study the relationship between the therapeutic effect of sericin on T2DM and the absorption of sericin by the body,five groups of animal experiments（Normal,SS-po,SS-ip,ad SS-ip,Model group）were designed to detect blood levels of sericin.The therapeutic effect of sericin concentration and different modes of administration on type 2 diabetes mellitus.The experimental results showed that（1）after oral and intraperitoneal injection of sericin,sericin could be detected in the blood,and the concentration was different,ad SS-ip（33.21±3.2132 ng/ml）>SS-ip（28.43±3.9009 ng/ml）>SS-po（17.05±2.9445 ng/ml）.（2）Sericin can effectively treat T2DM,and the FBG levels in the SS-po,SS-ip,ad SS-ip groups were significantly lower than those in the Model group at the fifth week of treatment.The hypoglycemic rate of the SS-ip group was 46.29%,the hypoglycemic rate of the ad SS-ip group was 41.94%,and the blood sugar level of the SS-po group was effectively controlled,which was always significantly lower than that of the Model group,and the hypoglycemic rate at the fourth week was 8.25%.（3）After sericin treatment,the insulin sensitivity of SS-po,SS-ip,and ad SS-ip groups was also slightly improved,and the three groups were improved by 7.62%,8.23%,and 5.29%respectively compared with the Model group;At the same time,insulin resistance was significantly improved,and the insulin resistance symptoms in the SS-po,SS-ip,and ad SS-ip groups were reduced by40.53%,42.78%,and 29.88%,respectively,compared with the Model group.In addition,glucose tolerance was also improved,and the area under the curve of glucose tolerance was reduced by 11.02%,56.15%,and 40.51%,respectively,compared with the model group.In addition,after sericin treatment,the arrangement of cells in liver,kidney and pancreas tissues was more regular,and the tissue damage was improved.The third part:Based on the Caco-2 cell model,the intestinal epithelial cell layer was simulated in vitro to study the absorption and transport of ad SS.The results found that oral sericin enters the blood and participates in the systemic circulation through passive transport and bypass transport.In this study,the Caco-2 cell model was used to study the absorption and transport of oral sericin.（1）The cytotoxicity experiment showed that when the concentration of ad SS was less than or equal to 1 mg/ml,it had no toxic effect on cells,and the cell viability was greater than 100%,set the ad SS concentration in transport experiments to 1 mg/ml.（2）Through microscope observation,detection of transmembrane resistance value,and detection of sodium fluorescein penetration,it was confirmed that the Caco-2 cell model was successfully constructed,and transmembrane transport and bypass transport were studied.The results showed that in the transmembrane transport,the transport amount of ad SS gradually accumulated and increased with time,and the cumulative transport amount of the AP→BL side（representing the transport from the AP side of the Caco-2 cell model to the BL side of the model）was greater than that of the BL→AP side（indicates transport from the BL side of the Caco-2 cell model to the AP side of the model）.In addition,the apparent permeability coefficient（Papp）was used to judge the absorption of the drug in the intestinal tract.The Papp values on the AP→BL side at 30min,60 min,90 min,120 min,and 150 min were:（1.5755±0.0859）×10-5cm·s-1,（0.991±0.0758）×10-5cm·s-1,（0.9883±0.0286）×10-5cm·s-1,（0.8984±0.0063）×10-5cm·s-1,（0.8373±0.0101）×10-5cm·s-1;the Papp values on the BL→AP side at 30 min,60 min,90min,120 min,and 150 min are:（1.1623±0.0573）×10-5cm·s-1,（0.8541±0.0488）×10-5cm·s-1,（0.7772±0.0168）×10-5cm·s-1,（0.6106±0.0097）×10-5cm·s-1,（0.5583±0.0152）×10-5cm·s-1.The Papp values are all greater than 1×10-6cm·s-1,indicating that ad SS has good absorption.The transport mode of ad SS can be judged by the excretion rate（ER）.It is between 0.5 and 1.5,indicating that ad SS is mainly passively transported in the Caco-2cell model,that is,the absorption and transport mode in the intestinal tract after oral administration of sericin is mainly passive transport and absorption.（4）The results of the alternative transport study showed that the addition of EDTA opened the tight junctions between cells,the transport amount of ad SS increased,and both the Papp value and the ER value increased.The Papp values of ad SS group and EDTA+ad SS group were,AP→BL side:（0.8381±0.04048）×10-5cm·s-1,（2.4409±0.16191）×10-5cm·s-1;BL→AP side:（0.5396±0.00253）×10-5cm·s-1,（2.8402±0.12649）×10-5cm·s-1.The ER values of the ad SS group and EDTA+ad SS group were 0.6438 and 1.1636,respectively,indicating that ad SS could be absorbed into the systemic circulation by way of bypass transport.In summary,after oral administration of sericin,it will be enzymatically hydrolyzed by gastrointestinal proteases,the molecule will become smaller,and absorbed into the blood by passive transport and bypass transport,participating in the systemic circulation,and exerting a therapeutic effect on diabetes,and the injection effect is more effective.excellent.This research clarified the absorption and transport of sericin in the gastrointestinal tract after oral administration,supplemented the basis for sericin in the treatment of diabetes,and provided support for further clinical application of sericin.In addition,the structure and sequence of sericin passing through the gastrointestinal barrier can be used for reference in oral protein drug protein carriers,and related research needs to be further in-depth. |
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