| Objective:Cancer pain remains one of the main symptoms in cancer patients.Presently,advancement towards early diagnosis in cancer therapy has been made,while the breakthrough of cancer pain is rather limited.Clinical evidence indicated that unrelieved cancer pain affected the quality of life and long-term survival.Clinical oncologists and physicians always get used to the opioids therapies along with serious side effects.Thus,researchers have been focused on the new therapy of cancer pain treatment.Traditional Chinese medicine has been considered as an important therapy in cancer management.Moreover,Chinese medicine has been widely applicated as an adjunct of "three ladder therapy" in the treatment of cancer pain.Previously,our research group found Chinese medicine alleviated cancer pain both directly by blocking TRPV1 signaling and indirectly by reducing tumor growth.Tian Chan capsule is originated from clinical practice under the therapeutic principle of Traditional Chinese medicine.We hereby further explore the effect and mechanism of Tian Chan capsule on the alleviation of cancer pain induced by sarcoma compression.Methods:(1)In vivo experiment:A murine model of tumor compression-induced pain was established,treated with different doses of Tian Chan capsule intervention and cinobufagin,detection of tumor volume,tumor weight,thermal hyperalgesia,mechanical allodynia.To observe the effect of Tian Chan capsule and cinobufagin on the tumor compression induced pain of sarcoma,the expression of TRPV 1,eIF2α,p-eIF2α,ERK and p-ERK was detected by Western Blot,the expression of TRPV 1,PERK and CHOP was detected by RT-PCR.We used Western Blot to detect the expression of p-ERK、p-mTOR、p-eIF2α、GFAP、IBA1 changes with the process of cancer pain.Furthermore,cancer pain-related behaviors,TRPV1.and endoplasmic reticulum stress signaling related protein expression were detected to observe the effects of mTOR inhibitors,eIF2α inhibitors,and Tian Chan capsule combine with inhibitors.(2)In vitro:We used S180 cell line,different concentration of Tian Chan capsule and the main component of cinobufagin were evaluated.The drug-related effects on proliferation rate,cell cycling,apoptosis,reactive oxygen species were detected using CCK8,cell cycle kit,apoptosis kit of AV/PI and reactive oxygen species.We used Western Blot to detect the effects of cinobufagin and different concentration of Tian Chan capsule on the expression of p-eIF2α and p-ERK after induction of total cell protein in S180.We used Western Blot to detect the effects of cinobufagin and different concentration of Tian Chan capsule on the expression of TRPV1,p-eIF2α,ERK and p-ERK after induction of total cell protein in TRPV1-transfected HEK 293 cells.Results(1)In vivo experiment1.A murine tumor compression model was established,Tian Chan capsule and cinobufagin were administered for 14 days.High dose of Tian Chan capsule exhibited an activity to decrease the volume and weight of tumor and no significant change was observed in cinobufagin and other doses of capsule treated groups.2.Compared with tumor group,high dose of Tian Chan capsule,tramadol and cinobufagin significantly suppressed the thermal hyperalgesia and mechanical allodynia at day 10 and day 14.HE staining and immunohistochemical detection indicated tumor compressed the sciatic nerve,while CD45+ positive cells were distributed around sciatic nerve.3.Immunohistochemical results suggested that high dose of Tian Chan capsule exhibited an activity to decrease the expression of CHOP protein compared with tumor group.Western Blot indicated that high dose of Tian Chan capsule and cinobufagin demonstrated a significant inhibition of p-ERK and p-eIF2α,while a medium dose of Tian Chan capsule could inhibit the expression of TRPV 1.Real-time PCR results showed that high dose capsule could inhibit the expression of TRPV 1 and CHOP genes.4.Compared with tumor group,rapamycin combine with capsule group significantly suppressed the thermal hyperalgesia and mechanical allodynia.5.At day 14 after inoculation of sarcoma,the expressions of GFAP,p-ERK,and p-mTOR in the spinal dorsal horn were remarkably increasing.Westen blots suggested Tian Chan capsule might acts synergistically with rapamycin to reduce the expressions of mTOR and GFAP,but not for IBA1 and p-ERK.6.After inoculation of sarcoma,the expressions of p-eIF2α in the spinal dorsal horn were remarkably increasing.Compared with vehicle group,the eIF2α inhibitor of salubrinal could reduce the expression of p-eIF2α.Furthermore,the administration of salubrinal from day 7 to day 9 once daily,was associated with less expressions of GFAP,p-ERK,and p-mTOR.These results indicated a cross-talk relationship between TRPV1 and endoplasmic reticulum stress signaling pathway.(2)In vitro experiment1.Compared with control group,Tian Chan capsule and cinobufagin demonstrated a dose-dependent manner on S180 cell line.The concentration of 1.25mg/ml and the above dose demonstrated a proliferation inhibition rate of more than 50%.48h calculation of the IC50 for Tian Chan capsule and cinobufagin is 0.759mg/ml and 131.6μM,respectively.2.Compared with control group,0.75 mg/ml of Tian Chan capsule significantly block S180 cells in S phase of the cell cycle with a proportion of 51.42%±2.86.0.375mg/ml,0.75 mg/ml and 1.5mg/ml Tian Chan capsule could reduce the G2/M phase proportion in S180 cells.No effects of Tian Chan capsule on sub G1 phase were observed.3.0.75 mg/ml and 1.5mg/ml of Tian Chan capsule demonstrated the induction effects of apoptosis on S180 cells.Hoechst staining indicated the effects of apoptosis caused by Tian Chan capsule and cinobufagin.4.Tian Chan capsule and 132μM cinobufagin lead to the increasing ROS accumulation on S180 cells.The presence of NAC could partially reverse the intracellular ROS accumulation caused by cinobufagin.5.Tian Chan capsule could increase the expression of p-eIF2α,while 1.5mg/ml capsule and 132μM cinobufagin were shown to inhibit the expression of p-ERK in S180 cells.Capsacin demonstrated a significant induction of increasing TRPV1 and p-eIF2α expression in TRPV 1-transfected HEK 293 cells.Tian Chan capsule showed a consistent inhibition on the expression of TRPV 1,p-ERK,and p-eIF2α in 293 cells.Compared with the control group,cinobufagin(132μM)group demonstrated the inhibition effects on TRPV1 and p-eIF2α,but not for p-ERK in 293 cells.Conclusion:In vivo studies confirm that Tian Chan capsule inhibited tumor growth and reduced tumor compression induced cancer pain.Tian Chan capsule exhibit analgesic effects might act through the regulation of cross-talk between TRPV1 and ER stress signaling pathway.In vitro studies confirm that Tian Chan capsule inhibited S180 cell growth,induced ROS accumulation,endoplasmic reticulum stress,finally leading to apoptosis.Tian Chan capsule inhibited the expression of TRPV1,p-ERK and p-eIF2α in TRPV1-transfected 293 cells. |
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