The Mechanism Of Berberine In Relieving Cisplatin-induced Peripheral Neuropathy

Objective:To investigate the effect of berberine on the inflammatory mechanism of cisplatin-induced peripheral neuropathy,and to determine whether TRPV1 receptor mediates the protection of berberine on peripheral nerves.To develop a new use of the traditional Chinese medicine berberine,and provide a theoretical basis for clinical treatment of chemotherapy-induced peripheral neuropathy.Method:In this study,it is observed that berberine can prevent CIPN induced by cisplatin(CDDP)both in vivo and in vitro.C57BL/6 and TRPV1 knockout mice were used.Western blot,immunofluorescence and real-time PCR,were used to detect the expression of inflammatory factors and TRPV1 in DRG cells.The platinum concentration in DNA was detected by inductively coupled plasma mass spectrometer(ICP-MS).Intracellular calcium ion was detected by calcium imaging.Enzyme-linked immunosorbent assay was used to detect the changes of inflammatory factors in serum.The length of axons was observed and measured by inverted fluorescence microscope system.Calcium imaging technique was used to detect intracellular calcium changes.DRG cell viability was measured by MTT assay.Apoptosis was detected by flow cytometry and TUNEL staining.Goals of this study were to explore the inflammatory mechanism of berberine in relieving cisplatin-induced peripheral neuropathy,and develop new uses of berberine.Moreover it will provide theoretical basis for the clinical treatment of CIPN.Results:1.Behavioral experiments showed that the heat pain threshold of mice began to decrease after two weeks of cisplatin administration,while the mechanical pain threshold decreased after there weeks.Berberine could alleviate pain behavior in all treatment groups.In the primary culture experiment of DRG neuron cells,the cell inhibition rate reached 40%-50%after incubation with 3-μM cisplatin(CDDP group)for forty-eight hours.Compared with the CDDP group,3-μM berberine incubated with 3-μM cisplatin for forty-eight hours could improve the cells viability of DRG and increase the axon length of neuron cells.These results suggested that berberine can improve cisplatin-induced peripheral nerve damage both in vivo and in vitro.2.After four weeks of cisplatin modeling,the binding rate of Pt-DNA was significantly higher than that of control group in DRG of C57BL/6 mice.Berberine can reduce the binding of Pt to DNA and inhibit DNA damage of neurons.The expression of TRPV1 in dorsal root ganglion was significantly increased in C57BL/6 mice injected with cisplatin for two and four weeks.Berberine can reduce the expression of TRPV1 in dorsal root ganglion,Especially 120 mg/kg berberine had the best improvement effect.After two weeks of treatment,berberine inhibited the high expression of NF-κB and JNK proteins and the low expression of pSTAT3,pJNK and pP38 proteins in dorsal root ganglion of C57BL/6 mice induced by cisplatin.After 4 weeks of treatment,berberine improved the inflammatory response of DRG mainly through promoting the expression of NF-κB and ERK protein and inhibiting the level of pJNK.Compared with CDDP group,berberine could decrease the levels of pro-inflammatory factor NF-κB,NGF,IL-1β,IL-6 and TNF-α in a dose-dependent manner and increase the level of anti-inflammatory factor IL-10 at two and four weeks in serum of mice.3.After TRPV1 gene was knocked out,the hyperalgesia induced by cisplatin was controlled within two weeks,but the threshold of thermal hyperalgesia was still decreased after three weeks.There had no effect on the cold pain threshold.It is suggested that berberine may be involved by blocking TRPV1 receptor 2 weeks ago,and other mechanisms may be involved after 3 weeks.Berberine can relieve pain allergy induced by cisplatin after 3 weeks.After TRPV1 gene was knockout,the expression of NF-κB in dorsal root ganglion did not change at two weeks in each group,but increased at four weeks in CDDP group.In TRPV1 knockout mice,cisplatin induced the expression of JNK,P38 and ERK 1/2 decreased,while the expression of pP38 and pERK1/2 increased in dorsal root ganglion at two weeks.At 4 weeks,the inflammatory signaling proteins of JNK/MAPK pathway were not changed in all groups.Compared with CDDP group,berberine could decrease the levels of pro-inflammatory factor NF-κB,NGF,IL-1β,IL-6 and TNF-α in a dose-dependent manner and increase the level of anti-inflammatory factor IL-10 at two and four weeks in serum of TRPV1 knockout mice.It is suggested that berberine can reduce the expression of pro-inflammatory factors in serum but not affected by TRPV1 deletion.4.Primary cultures of DRG neurons showed that the early apoptosis of DRG neurons was mainly inhibited by co-incubation of berberine and cisplatin for twenty-four hours,late apoptosis was mainly inhibited by incubation for forty-eight hours.The expression of NF-κB,JNK,P38 and pP38 increased and the expression of pJNK decreased in DRG neurons incubated with 3μM cisplatin for 48 hours.Co-incubation with 3μM berberine inhibited the expression of NF-κB and P38.Calcium imaging results showed that berberine could inhibit the increase of intracellular calcium concentration triggered by cisplatin.Conclusion:Berberine can alleviate cisplatin-induced peripheral nerve pain in vivo and in vitro.In early stage,berberine may block TRPV1 to influence intracellular calcium concentration,and inhibite of the expression of NF-κB protein in DRG,and then regulate JNK/MAPK signaling pathway.Berberine decreased the expression of proinflammatory factors in serum,but this phenomenon may not be related to TRPV1.Berberine may also exert an anti-inflammatory effect through a mechanism other than TRPV1,to inhibit systemic inflammatory response caused by cisplatin.