Effects Of Jiawei Guizhi Fuling Pill On The Expression Of LC3, P62 And Beclin-1 In Sciatic Nerve Of PDPN Rat

Purpose:In this experiment,we investigated the effects of the Chinese herbal formula Gui Zhi Fu Ling Wan on the expression of LC3,P62 and Beclin-1 in the sciatic nerve tissue of rats with painful diabetic peripheral neuropathy(PDPN).Material and method:Using the random number table method,104 SPF-rated male SD rats,12 of which were randomly selected as the blank control(CON)group,were kept on a general diet throughout.The remaining 92 rats were fasted for 12 hours and then injected intraperitoneally with 2% streptozotocin(STZ)solution at 50 mg/kg.72 hours later,rats with tail vein glucose concentration ≥ 16.7 mmol/L were included in the follow-up study.Rats with blood glucose concentrations below 16.7 mmol/L were excluded.mechanical and thermal pain thresholds were measured after 4 weeks,and rats with a ≥50% decrease in mechanical or thermal pain thresholds were considered to be successfully modelled with PDPN.The rats were randomly divided into 5 groups and administered by gavage for 8 weeks.The groups and doses were as follows: model(P)group,0.9% Na Cl solution 10 ml/kg/d;plus-flavoured Gui Zhi Fu Ling Wan low dose(L)group,9.0 g/kg/d of Chinese herbal soup;plus-flavoured Gui Zhi Fu Ling Wan medium dose(M)group,18.0 g/kg/d of Chinese herbal soup;plus-flavoured Gui Zhi Fu Ling Wan high dose(H)group,36.0 g/kg/d of Chinese herbal soup.The rats were tested for general condition,blood glucose,body weight,mechanical pain threshold,thermal pain threshold and nerve conduction velocity at the end of week 0,2,4,6 and 8 after administration.The sciatic nerve was stained with HE and the expression of LC3,P62 and Beclin-1 in the sciatic nerve was measured by Real-time RTq-pcr and Western Blot.The data were statistically analysed using Graph Pad Prism 9.0.0 software and histograms were plotted at P<0.05.Results:1.The rats in the model(P)group were in poor mental condition and showed symptoms of excessive drinking,excessive eating,excessive urination and wasting,withered and lusterless fur,and symptoms such as curled up and arched back,slow movement and heavy odour;the rats in the low(L),medium(M)and high(H)dose groups of TCM and the methylcobalamin(J)group also showed the above symptoms,but to a lesser extent than the model(P)group.The improvement of the above symptoms was relatively obvious in the high-dose group.2.The body weight level of the model(P)group was significantly lower than that of the blank control(CON)group(P<0.01);the body weight level of the Chinese medicine(L),medium(M)and high dose(H)groups and the mecobalamin(J)group increased significantly at 4 and 8 weeks compared with that of the model(P)group(P<0.05).3.The blood glucose levels in the model(P)group were significantly increased compared with those in the blank control(CON)group(P<0.01);the blood glucose levels in the Chinese medicine(L),medium(M)and high dose(H)groups and the mecobalamin(J)group were significantly decreased at 2,4 and 8 weeks compared with those in the model(P)group(P<0.01).4.The thermal and mechanical pain thresholds in the model(P)group were significantly lower compared with the blank control(CON)group(P<0.01);the thermal and mechanical pain thresholds in the Chinese medicine(L),medium(M)and high dose(H)groups and the mecobalamin(J)group were significantly higher at 4 and 8 weeks compared with the model(P)group(P<0.01).5.The nerve conduction velocity levels in the model(P)group were significantly lower compared with those in the blank control(CON)group(P<0.01);the nerve conduction velocity levels in the Chinese herbal medicine(L),medium(M)and high dose(H)groups and the mecobalamin(J)group at 2,4 and 8 weeks were significantly higher compared with those in the model(P)group(P<0.01).6.The results of HE staining of the sciatic nerve tissues of the rats in each group showed that,compared with the blank control(CON),the nerve fibers in the model(P)group were disorganized,of different sizes and incomplete morphology,with obvious pathological changes,a large number of nerve fibers were edematous,and the myelin sheath structure was lost and vacuolated;compared with the model(P),the Chinese medicine group and the mecobalamin(J)group were more regular in arrangement,more uniform in morphology,with a higher degree of myelination.Compared with the model(P),the Chinese herbal medicine and methylcobalamin(J)groups had more regular arrangement,more uniform morphology,higher myelination and less edema,among which the high-dose(H)group with Gui Zhi Fu Ling Wan was slightly better than the other treatment groups,and the orange petal-like structures in the former were clearer and more uniformly arranged than the latter.7.The results of Real-time RTq-pcr and Western blot showed that compared with the blank control(CON)group,the expression levels of LC3 and Beclin-1 in the sciatic nerve tissues of the remaining groups were significantly lower and the expression level of P62 was higher,and the differences were statistically significant(P < 0.01).Compared with the model(P)group,the expression levels of LC3 and Beclin-1 in the sciatic nerve tissues of rats in the Chinese herbal medicine(L),medium(M)and high dose(H)groups and the methylcobalamin(J)group were significantly increased and the expression level of P62 was decreased,and the differences were statistically significant(P<0.01).Conclusion:1.In this experiment,a diabetic model was established by intraperitoneal injection of streptozotocin(STZ)solution,and the model was screened by a 50% decrease in mechanical pain threshold or thermal pain threshold after 4 weeks.The results demonstrated that although the modeling period was longer,the method was more able to restore the evolution from diabetes to PDPN,and the modeling rate was 80.43 %.2.The Chinese herbal compound Gui Zhi Fu Ling Wan can improve the mental status and behavioral changes of PDPN rats.3.The Chinese herbal formula with Gui Zhi Fu Ling Wan could improve the body weight,blood glucose,pain threshold and nerve conduction velocity of PDPN rats.4.The Chinese herbal formula with Gui Zhi Fu Ling Wan could improve the degree of sciatic nerve injury in PDPN rats.5.The improvement of sciatic nerve in PDPN rats by the Chinese medicine compound Gui Zhi Fu Ling Wan may be achieved by up-regulating the levels of LC3 and Becli n-1 and down-regulating the horizontal expression of P62.