Isolation And Identification Of Peptides From Panacis Quinquefolii Radix And Their Ameliorative Effect On Traumatic Skin Injury In Mice

Background: As the body’s first line of defence against the environment,the skin is vulnerable to damage from the external environment.The presence of skin damage affects people’s daily life.American ginseng(Panax quinquefolium L.)is one of the advantageous characteristic Chinese herbs in Jilin Province and one of the commonly used bulk herbs in China,which is widely used in health care products and pharmaceuticals.Despite the diverse activities of the peptide-like components of Panax quinquefolium,there are relatively few relevant studies.Methods: 1.Solvent extraction and acetone precipitation were used to obtain the western ginseng peptides,and the extraction process was optimized using response surface screening.Further,the separation was purified using Sephadex G-25 chromatography,and the isolated fractions were screened for activity.Finally,the best fractions were identified by molecular weight and sequence using Tricine-SDS-PAGE and mass spectrometry.2.A mouse injury model was established and the skin pathological changes were observed by H&E staining and Masson’s trichrome staining test.The pharmacological effects of PGP were further investigated using enzyme-linked immunosorbent assay(ELISA),immunohistochemical staining and immunoblotting(Western Blot).3.In vitro models of H2O2-induced damage to HUVECs and HACAT cells were established.Cell proliferation and toxicity assays were performed to screen the optimal PGP administration concentration.The inhibitory effect of PGP on apoptosis was evaluated using Hoechst 33258 staining,ROS staining,cell mitochondrial membrane potential(JC-1)assay,immunofluorescence staining and Western Blot.Finally,Western Blot assay was used to investigate the mechanism of PGP amelioration of inflammation in endothelial and keratinocytes.Results: 1.The optimal process conditions for PGP extraction were screened based on the experimental results of response surface,and the predicted extraction rate of American ginseng was 23.41±0.23%.The results of Tricine-SDS-PAGE and mass spectrometry showed that the best F2 fraction for combined activity consisted of 17 fractions with different relative molecular weights and sequence distribution between 764-2390 Da,and 79 eligible peptides were screened.2.PGP may inhibit the oxidative stress response by 2.PGP may have a reparative effect on mouse wounds by inhibiting oxidative stress and reducing the occurrence of inflammatory phenomena.Further Western Blot studies showed that PGP inhibited apoptosis in skin tissues through the PI3K/AKT pathway and was involved in angiogenesis,thereby promoting skin healing.3.PGP reduced the rate of apoptosis in a dose-dependent manner,decreased abnormal expression of intracellular ROS and improved mitochondrial transmembrane potential depolarization.4.Blot assays showed that PGP could reduce apoptosis in HUVECs and HACAT cells through the PI3K/AKT pathway.Meanwhile,PGP could inhibit inflammation through the NF-κB pathway.In addition,PGP could play a role in promoting skin wound healing by regulating angiogenesis-related factors in HUVECs and HACAT cells,and thus in skin wound healing.Conclusion: We explored the process optimization of PGP and further characterized the molecular weight and sequence of the screened active fractions.Through the establishment of in vivo and in vitro model experiments,we found that PGP could improve the damaged skin state,inhibit the oxidative stress and inflammatory response generated after skin damage,as well as regulate the angiogenic process.This study expands the application field of western ginseng peptides and provides a new idea for the development of skin therapeutic drugs.